1This study was designed to establish the basis for altered membrane excitability during the inhibition of mitochondrial metabolism in central mammalian neurons. Perforated whole‐cell patch clamp and fluorimetric techniques were combined to examine changes in membrane currents, intracellular calcium ([Ca2+]i) and mitochondrial potential (ΔΨm) in neurons dissociated from the CA1 subfield of the hippocampus of young rats.2On application of the mitochondrial inhibitor NaCN, or the uncoupler carbonyl cyanide p‐trifluoromethoxyphenylhydrazone (FCCP), the membrane potential hyperpolarized and membrane conductance increased. Under voltage clamp, an outward current was seen. The reversal potential of the current at −83 mV and its dependence on extracellular [K+] confirmed that this was a K+conductance.3Simultaneous recordings of [Ca2+]iand current showed a striking correlation between a rise in [Ca2+]iand the developed outward current. Flash photolysis of the caged Ca2+chelator, diazo‐2, reversed both the rise in [Ca2+]iand the outward current. The current was reduced by 80% by charybdotoxin, was attenuated by 10 mmTEA+but was unaffected by apamin or by the KATPchannel blocker tolbutamide (400 μm−1 mm). These data suggest strongly that the current is carried by Ca2+‐dependent K+channels.4Simultaneous recordings of membrane current, ΔΨmand [Ca2+]irevealed the sequence of events in response to impaired mitochondrial function (CN, FCCP or anoxia): ΔΨmdepolarized, followed rapidly by an increase in [Ca2+]ifollowed in turn by the outward current. [Ca2+]iand membrane current recovered only after mitochondrial repolarization.5The rise in [Ca2+]iappeared to result from an increased Ca2+influx through voltage‐gated Ca2+channels. It was dependent on extracellular Ca2+and was much reduced by methoxyverapamil (D600). The rate of Mn2+quench of fura‐2 fluorescence was increased by the inhibitors, and the inhibitors induced a small inward current when K+channels were blocked that preceded the rise in [Ca2+]i. However, the increase in [Ca2+]ishowed no obvious dependence on membrane potential in cells clamped at a range of holding potentials from −90 to −45 mV.6Thus, removal of oxygen, uncoupling mitochondrial oxidative phosphorylation or inhibition of respiration, all lead to mitochondrial depolarization, an increased Ca2+influx through (voltage‐gated) channels, even at hyperpolarized membrane potentials, raising [Ca2+]iwhich in turn drives an increased K+conductance that modulates membrane excitability.