The endothelium-dependent hyperpolarization of cells has a crucial role in regulating vascular tone, especially in microvessels. Nitric oxide (NO) and prostacyclin (PGI₂), in addition to endothelium-derived hyperpolarizing factor (EDHF), have been reported to hyperpolarize vascular smooth muscle in several organs. The hyperpolarizing effects of these factors have been reported to be increased by a stretch in large coronary arteries. EDHF has not yet been identified and cytochrome P450 metabolites and hydrogen peroxide (H₂O₂) are candidates for EDHF. Using a membrane potential-sensitive fluorescent dye, bis-(1,3-dibutylbarbituric acid)trimethione oxonol (DiBAC₄(3)), we examined whether NO, PGI₂, cytochrome P450 metabolites and H₂O₂ contribute to acetylcholine (ACh)-induced hyperpolarization in pressurized coronary microvessels. Canine coronary arterial microvessels (internal diameter, 60 to 356 µm) were cannulated and pressurized at 60 cmH₂O in a vessel chamber perfused with physiological salt solution containing DiBAC₄(3). Fluorescence intensity and diameter were measured on a computer. There was a linear correlation between changes in the fluorescence intensity and membrane potential. ACh significantly decreased the fluorescence intensity (hyperpolarization) of the microvessels without any inhibitors. Endothelial damage caused by air perfusion abolished the ACh-induced decrease in fluorescence intensity. Inhibitiors of NO synthase and cyclooxygenase did not affect the ACh-induced decreases in the fluorescence intensity. The addition of 17-octadecynoic acid (cytochrome P450 monooxygenase inhibitor) to those inhibitors significantly attenuated the ACh-induced decreases in fluorescence intensity, whereas catalase (an enzyme that dismutates H₂O₂ to form water and oxygen) did not. Furthermore, catalase did not affect the vasodilation produced by ACh. These results indicate that NO and PGI₂ do not contribute to the ACh-induced hyperpolarization and that the cytochrome P450 metabolites but not H₂O₂ are involved in EDHF-mediated hyperpolarization in canine coronary arterial microvessels.