1. The influence of different inhibitors of cytochrome P450 mono-oxygenase on the endothelium-dependent and -independent hyperpolarization in the isolated rat main mesenteric artery was investigated. 2. Application of acetylcholine (ACh; 1 microM) for 10 min evoked an endothelium-dependent peak hyperpolarization of about 18 mV followed by a partial recovery to a level 7 mV more negative than the resting value (-50.2 +/- 0.5 mV). 3. Proadifen (30 microM) completely and reversibly inhibited the ACh-induced hyperpolarization. Conversely, the imidazole antimycotics clotrimazole (30 microM) and miconazole (100 microM) had less effect on the peak endothelium-dependent hyperpolarization. The suicide substrate inhibitors 17-octadecynoic acid (17-ODYA; 5 microM) and 1-aminobenzotriazole (1-ABT; 2 mM) did not significantly influence endothelium-dependent hyperpolarization. 4. The endothelium-independent hyperpolarization (16 mV) evoked by leveromakalim (300 nM) was completely inhibited by proadifen as well as by clotrimazole and miconazole but was not affected by 17-ODYA or 1-ABT. 5. These results do not support the view that the ACh-induced endothelium-dependent hyperpolarization in the rat mesenteric artery is mediated by cytochrome P450 mono-oxygenase metabolites. Proadifen and imidazole antimycotics impair the activation of ATP-regulated K+ channels in mesenteric artery cells, rendering non-specific inhibition of smooth muscle K+ channel activation an alternative explanation for the inhibitory influence of some (but not all) P450 inhibitors on endothelium-dependent hyperpolarization in this preparation.