1. Many endothelium-dependent vasodilators hyperpolarize the endothelial cells in blood vessels. It is not known whether these hyperpolarizations are linked to nitric oxide synthesis or to an endothelium-derived hyperpolarizing phenomenon, since most of the vasodilators release both factors. In this context, we first verified that the endothelium-dependent relaxations induced by 5-hydroxytryptamine (5-HT) on pig coronary arteries are due only to the activation of the nitric oxide pathway. Then we studied the effects of 5-HT on membrane potential of endothelial and smooth muscle cells. 2. In the absence of endothelium, 5-HT caused a concentration-dependent contraction of coronary artery strips. No change of the smooth muscle cell membrane potential was observed during contraction to 1 microM 5-HT. 3. In the presence of 1 microM ketanserin to suppress the contractile effect of 5-HT, 5-HT induced concentration-dependent relaxation of endothelium-intact strips precontracted by 10 microM prostaglandin F2 alpha (PGF2 alpha). These relaxations were suppressed by 1 microM NG-nitro-L-arginine, an inhibitor of nitric oxide synthesis, showing that they were produced predominantly by nitric oxide. 4. In the presence of 1 microM ketanserin, 1 microM 5-HT did not change the smooth muscle cell membrane potential of strips precontracted by either 10 microM PGF2 alpha or by 10 microM acetylcholine (ACh). In the same conditions, 1 microM 5-HT caused a weak 2.6 +/- 0.4 mV hyperpolarization, of the endothelial cells. 5. In conclusion, the fact that 5-HT did not change the membrane potential of smooth muscle cells and only weakly hyperpolarized the endothelial cells during relaxations, suggests that in both cell types no electrical events accompany activation of the nitric oxide pathway. This is in contrast to the hyperpolarizations observed in endothelial and smooth muscle cells when the endothelium-derived hyperpolarization factor (EDHF) pathway is activated.