The L-type Ca(2+) channel is responsible for initiating contraction in the heart. Mitochondria are responsible for meeting the cellular energy demands and calcium is required for the activity of metabolic intermediates. We examined whether activation of the L-type Ca(2+) channel alone is sufficient to alter mitochondrial function. The channel was activated directly with the dihydropyridine agonist BayK(-) or voltage-clamp of the plasma membrane and indirectly by depolarization of the membrane with high KCl. Activation of the channel increased superoxide production (assessed as changes in dihydroethidium fluorescence), NADH production and metabolic activity (assessed as formation of formazan from tetrazolium) in a calcium-dependent manner. Activation of the channel also increased mitochondrial membrane potential assessed as changes in JC-1 fluorescence. The response was reversible upon inactivation of the channel during voltage-clamp of the plasma membrane and did not appear to require calcium. We examined whether the response may be mediated through movement of cytoskeletal proteins. Depolymerization of actin or exposing cells to a peptide directed against the alpha-interacting domain of the alpha(1C)-subunit of the channel (thereby preventing movement of the beta-subunit) attenuated the increase in mitochondrial membrane potential. We conclude that activation of the L-type Ca(2+) channel can regulate mitochondrial function and the response appears to be modulated by movement through the cytoskeleton.