Ca2+ uptake by mitochondria might both modulate the cytosolic Ca2+ concentration ([Ca2+]c) and depolarize the mitochondrial membrane potential (ΔΨm) to limit ATP production. To investigate how physiological Ca2+ signaling might affect energy production, ΔΨm was examined during Ca2+ oscillations in smooth muscle cells. In single, voltage-clamped smooth muscle cells, inhibition of mitochondrial Ca2+ accumulation inhibited inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3]-evoked Ca2+ release and prolonged the time required for restoration of [Ca2+]c following activation of plasmalemmal Ca2+ currents (ICa). Ca2+ could be released from mitochondria immediately (within 15 seconds) after a [Ca2+]c rise evoked by Ins(1,4,5)P3 or ICa. Despite this evidence of mitochondrial Ca2+ accumulation, no change in ΔΨm was observed during single or repetitive [Ca2+]c oscillations evoked by these conditions. Occasionally, spontaneous, repetitive, persistent Ca2+ oscillations were observed. In these cases, mitochondria displayed stochastic ΔΨm depolarizations, which were independent both of events in neighboring mitochondria and of the timing of the [Ca2+]c oscillations themselves. Such ΔΨm depolarizations could be mimicked by increased exposure to either fluorescence excitation light or the ΔΨm-sensitive dye tetramethylrhodamine ethyl ester (TMRE) and were inhibited by antioxidants (ascorbic acid, catalase, Trolox and TEMPO) or the mitochondrial permeability transition pore (mPTP)-inhibitor cyclosporin A (CsA). Individual mitochondria within smooth muscle cells might depolarize during repetitive Ca2+ oscillations or during oxidative stress but not during the course of single [Ca2+]c transients evoked by Ca2+ influx or store release.