We have studied the effects of cytochrome P450 inhibitors on the entry of Ca2+ and Mn2+, used here as a Ca2+ surrogate for Ca2+ channels, in fura-2-loaded GH3 pituitary cells and bovine chromaffin cells depolarized with high-K+ solutions. Imidazole antimycotics were potent inhibitors (econazole greater than miconazole greater than clotrimazole greater than ketoconazole). alpha-Naphtoflavone and isosafrole, but not metyrapone, also inhibited the entry of Ca2+ and Mn2+ induced by depolarization. This inhibitory profile most resembles that reported for IA-type cytochrome P450. However, carbon monoxide (CO), a well-known cytochrome P450 antagonist, had no effect on Ca2+ (Mn2+) entry. Given the high selectivity of the imidazole antimycotics for the heme moiety, our results suggest that a hemoprotein closely related to cytochrome P450 (but insensitive to CO) might be involved in the regulation of voltage-gated Ca2+ channels. The inhibitory pattern was also similar to that previously reported for agonist-induced Ca2+ (Mn2+) influx in neutrophils and platelets, although CO was an efficient inhibitor in this case. These results pose the question of whether similarities in the sensitivity to cytochrome P450 inhibitors exhibited by receptor-operated and voltage-gated channels reflect unknown similarities either in structural features or regulation mechanisms.