The mitochondrial membrane potential (DeltaPsi(m)) underlies many mitochondrial functions, including Ca(2+) influx into the mitochondria, which allows them to serve as buffers of intracellular Ca(2+). Spontaneous depolarizations of DeltaPsi(m), flickers, have been observed in isolated mitochondria and intact cells using the fluorescent cationic lipophile tetramethylrhodamine ethyl ester (TMRE), which distributes across the inner mitochondrial membrane in accordance with the Nernst equation. Flickers in cardiomyocytes have been attributed to uptake of Ca(2+) released from the sarcoplasmic reticulum (SR) via ryanodine receptors in focal transients called Ca(2+) sparks. We have shown previously that an increase in global Ca(2+) in smooth muscle cells causes an increase in mitochondrial Ca(2+) and depolarization of DeltaPsi(m). Here we sought to determine whether flickers in smooth muscle cells are caused by uptake of Ca(2+) released focally in Ca(2+) sparks. High-speed three-dimensional imaging was used to monitor DeltaPsi(m) in freshly dissociated myocytes from toad stomach that were simultaneously voltage clamped at 0 mV to ensure the cytosolic TMRE concentration was constant and equal to the low level in the bath (2.5 nM). This approach allows quantitative analysis of flickers as we have previously demonstrated. Depletion of SR Ca(2+) not only failed to eliminate flickers but rather increased their magnitude and frequency somewhat. Flickers were not altered in magnitude or frequency by ryanodine or xestospongin C, inhibitors of intracellular Ca(2+) release, or by cyclosporin A, an inhibitor of the permeability transition pore. Focal Ca(2+) release from the SR does not cause flickers in the cells employed here.