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By means of the fura-2 technique and image analysis the intracellular concentration of free calcium ions [Ca 2+ ] i was examined in isolated rainbow trout pinealocytes identified by S-antigen immunocytochemistry. Approximately 30% of the pinealocytes exhibited spontaneous [Ca 2+ ] i oscillations whose frequency differed from cell to cell. Neither illumination with bright light nor dark adaptation of the cells had an apparent effect on the oscillations. Removal of extracellular Ca 2+ or application of 10 μ M nifedipine caused a reversible breakdown of the [Ca 2+ ] i oscillations. Application of 60 mM KCl elevated [Ca 2+ ] i in 90% of the oscillating and 50% of the non-oscillating pinealocytes. The effect of KCl was blocked by 50 μ M nifedipine. These results suggest that voltage-gated L-type calcium channels play a major role in the regulation of [Ca 2+ ] i in trout pinealocytes. Experiments with thapsigargin (2 μ M) revealed the presence of intracellular calcium stores in 80% of the trout pinealocytes, but their role for regulation of [Ca 2+ ] i remains elusive. Treatment with norepinephrine (100 pM–50 μ M), previously shown to induce calcium release from intracellular calcium stores in rat pinealocytes, had no apparent effect on [Ca 2+ ] i in any trout pinealocyte. This finding conforms to the concept that noradrenergic mechanisms are not involved in signal transduction in the directly light-sensitive pineal organ of anamniotic vertebrates.