An analytical study of the cyclic stomatal behaviour in leaves of bean, Phaseolus vulgaris L. 'Vroege Wagenaar', was made in order to explore the cycling, to study different aspects of plant water relations and of the action mechanism of the stomata. A general method of investigation was recording of the difference of temperature of leaf and air with thermocouples in a constant environment.Introductory observationsPlants grown at low light intensity showed a higher tendency to sustained cycling (higher instability of the stomatal apparatus) than those grown at higher light intensity. At the higher light intensity non-aeration of the nutrient solution increased the tendency to cycling with a more than additive effect. It is suggested that the lower capacity to take up water of non-aerated roots was involved in the higher tendency to cycling. Cycling over the whole plant was frequently disturbed by bringing cycling of a part of the active stomata out of phase. Cycling was induced by changing abruptly any factor that affected stomatal opening, by entrainment with cycling air vapour pressure, by entrainment with cycling plant water potential, and apparently spontaneously.Younger leaves showed shorter free-running periods than older ones. When both were attached on the same plant, cycling in the younger leaves entrained cycling in the older ones.Water relations of cyclingAmplitudes of cycling increased by decreasing air humidity and by increasing the water potential in the root medium. A time delay in the adjustment of the turgor of the guard cells to the plant water potential is assigned to be the cause of overshooting of the active stomatal movement and as to be the factor amplifying both passive opening and closing movement. Cycling only occurred when an important resistance to water uptake was present in the transpiration stream. In intact plants this resistance was located in the roots. The water potential cycled in the whole plant down to this resistance, synchronizing cycling in the whole plant. During each cycle the root resistance increased during the sub-period with low rates of water uptake and decreased delayed at subsequently decreasing plant water potential. Due to the fact that the fall of root resistance was enhanced by increasing root temperature, both cycling of stomatal opening and cycling of root resistance were intensified.A diagrammatic conceptual model is presented, showing the pathway of the transpiration stream, of the transfer of water within the leaf, and of the stomatal control system of the water balance of the leaf. A negative feedback control circuit in it indicates how active stomatal movements function, while a positive feedback circuit shows how passive stomatal movements function during cycling. Circadian rhythm in steady and cycling stomatal openingsIn continuous light a circadian rhythm in non-cycling stomatal opening was found, which phase could be caused to shift. Cycling was most intense at the time that in similarly treated plants the maximum was attained in the circadian rhythm in the opening in non-cycling stomata and least intense half a circadian period later. In continuous darkness during the first day after the last photoperiod, stomata opened in a cyclic way with a daily pattern in the amplitudes as well as in the number of cycles. During photoperiods of 17 hours the noncycling stomatal opening in leaves, attached to plants with and without root systems, as well as in isolated leaves followed a daily pattern. During these photoperiods the modulation of amplitudes and periods showed the same daily pattern as during the circadian rhythm in continuous light. During the daily dark periods stomata remained closed. A daily pattern was observed in the onset of stomata] opening after short dark periods in the course of the daily photoperiod. The data suggest that one endogenous circadian oscillator, situated in the leaves, caused the circadian rhythm in non-cycling stomatal opening and modulated stomatal cycling in continuous light, continuous darkness and during the daily photoperiods.Environmental effects on cycling stomataThe time between the onset of light and the beginning of stomatal opening increased with increasing length of the. preceding dark period at equal phases of the circadian rhythm. After 1 hour in the light the effect of the preceding dark period had almost disappeared. After removal of the trifoliates the periods in the primary leaves gradually shortened in the course of some days. It is suggested that this increased activity was caused by a reduced competition for minerals, enzymes and proenzymes from the root.Cycling was frequently damped by increasing the irradiation intensity. Irradiation intensity beyond 34,000 erg cm -2sec -1affected the rate of CO 2 exchange, but not the period or amplitude of sustained cycling. When the irradiation intensity on the lower surface of the leaf during the diurnal photoperiod was decreased from 2100 to 800 erg cm -2sec -1, cycling continued. The amplitude decreased and the period increased, mainly due to the increase of the sub-period with minimal stomatal opening. During the photoperiod 150 erg cm -2sec -1on the upper surface of the leaf induced overshooting stomatal opening. The sub-period with minimal stomatal opening, being similar to STÅLFELT'S 'Spannungsphase' (build-up period), increased at decreasing irradiation intensity due to an increase in time required for the guard cells to exceed the counterpressure from the surrounding cells, hence, the product rule of STÅLFELT applied here.The build-up period of stomatal opening increased by decreasing the CO 2 -concentration of the air from 300 to 200 to 0 ppm.Enhancing the temperature in light decreased the period. This effect was clearly distinguished from the effect of increasing vapour pressure gradient. The Q 10 of decrease fell gradually from approximately 4 between 17° and 20°C to approximately 1.5 between 25° and 29°C. The sub-period with minimal stomatal opening was mainly affected. In light the same effect of temperature on cycling was found in CO 2 -free air as in normal air and the same on cycling in detached leaves as in attached leaves. A temperature increase in darkness induced stomatal cycling; this was assigned for the major part to the stimulation of the active opening component. Temperature is concluded to exert an influence independent of the effect of CO 2 -concentration and of the water balance.The effects of light and temperature on cycling support the existing hypothesis for an active ATP using potassium pump as a mechanism for stomatal opening.Conclusive remarksSeveral internal plant factors play a part in causing instability of the stomatal apparatus, but also the habit of the plant. Cycling must be expected to occur in the field and in the glasshouse, especially at low air humidity, when these factors favour instability. Severe stomatal cycling is suggested to decrease mean assimilation rate.