The mechanism of the sustained acetylcholine-induced endothelium-dependent hyperpolarization (EDH) in intact rat small mesenteric arteries prestimulated with noradrenaline (10-6 mol/l) was investigated by means of the single microelectrode voltage clamp method. The vascular smooth muscle cells (VSMCs) in this preparation are not coupled. Sustained application of acetylcholine (ACh ,10-5 mol/l, > 5 min) hyperpolarized the VSMCs by induction of a hyperpolarizing current. This effect was completely blocked by the inhibitor of the nitric oxide (NO) synthase L-NAME (Nω-nitro-L-arginin-methylester, (10-3 mol/l) but not by the inhibitor of the soluble guanylate cyclase (sGCl), methylene blue (MB, 10-4 mol/l). Application of the NO-donor sodium nitroprusside (SNP, 10-6 mol/l) for more than 5 min mimicked the induction of the endothelium-dependent hyperpolarizing current in vessels with destroyed endothelium. The reversal potential of this current is dependent on the extracellular K+ concentration. The effect of SNP could also not be blocked by MB. The blockers of ATP-dependent and Ca2+-dependent K+ channels, glibenclamide (Glib, 10-5 mol/l) and charybdotoxin (CTX, 5x10-8 mol/l), respectively, blocked a hyperpolarizing current in the VSMCs similar to the ACh- or SNP-induced current. The isolated application of either Glib or CTX did not block the activation of the hyperpolarizing current by SNP. Only the combined administration of Glib and CTX blocked the SNP-induced current completely. Our results suggest that in rat small mesenteric artery, ACh hyperpolarizes the VSMCs tonically by activating both ATP- and Ca2+-dependent K+ currents only via release of NO from the endothelium without need for activation of the sGCl.