The mitochondrial membrane potential (_m) underlies many mitochondrial functions, including Ca²⁺ influx into the mitochondria, which allows them to serve as buffers of intracellular Ca²⁺. Spontaneous depolarizations of _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 Ca²⁺ released from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) in focal transients called Ca²⁺ sparks. We have shown previously that an increase in global Ca²⁺ in smooth muscle cells causes an increase in mitochondrial Ca²⁺ and depolarization of _m. Here we sought to determine whether flickers in smooth muscle cells are caused by uptake of Ca²⁺ released focally in Ca²⁺ sparks. High-speed 3D imaging was used to monitor _m in freshly dissociated myocytes from toad stomach which were simultaneously voltage clamped at 0 mV to ensure the cytosolic [TMRE] ([TMRE]_c) 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 Ca²⁺ 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 Ca²⁺ release, nor by Cyclosporin A, an inhibitor of the permeability transition pore (PTP). Focal Ca²⁺ release from the SR does not cause flickers in the cells employed here.