Rotenone (Rot) is a plant toxin that stimulates superoxide (O2−) release from complex I of mitochondrial respiratory chain. It is used as a pharmacological tool to study roles of O2− in redox-related pathophysiology represented by Parkinson's disease. However, its effects on ion channel currents have little been elucidated. To study O2−-dependent regulation of L-type Ca2+ channel current (ICa,L) we examined acute effect of Rot on ICa,L in cultured A7r5 cells derived from embryonic rat aorta. ICa,L was recorded by ruptured whole-cell clamp technique with 10 mM Ba2+ as a charge carrier with high resistance pipettes that minimized run-down of the current. Rot dose-dependently (10nM-0.01mM) increased ICa,L. The increase was transient and was followed by the decrease of the amplitude and increase of voltage-dependent inactivation. It occurred with a characteristic shift of I-V relationship to the left and succeeding inhibition was accompanied with a shift of steady-state inactivation curve to the left. Dithiothreitol (DTT) induced similar increase of ICa,L but without any negative shift. Rot produced additional increase of ICa,L with the negative shift in the presence of DTT. Sustained pretreatment by1mM-tempol, scavenger of O2−, did not decrease basal ICa,L density but suppressed the Rot-induced increase of ICa,L. Since O2− is precursor of H2O2, we examined involvement H2O2 on the Rot-induced modulation of ICa,L. H2O2 (0.3 mM) significantly inhibited ICa,L. Also in the presence of H2O2, Rot increased ICa,L with a negative shift of I-V relationship and the increase diminished with time. We conclude that rotenone-induced augmented release of O2− from mitochondrial complex I increased ICa,L and the modulation is independent of H2O2 in A7r5 aortic smooth muscle cells. O2− may be involved in the regulation of basal ICa,L.