Activation of both small-conductance (SKCa) and intermediate-conductance (IKCa) Ca2+-activated K- channels in endothelial cells leads to vascular smooth muscle hyperpolarization and relaxation in rat mesenteric arteries. The contribution that each endothelial K-channel type makes to the smooth muscle hyperpolarization is unknown. In the presence of an NO synthase inhibitor, ACh evoked endothelium and concentration-dependent smooth muscle hyperpolarization, increasing the resting potential (~ -53 mV) by around 20 mV at 3 µM. Similar hyperpolarization was evoked with cyclopiazonic acid (SERCA inhibitor, 10 µM CPA), while 1- EBIO (300 µM, IKCa activator) only increased the potential by a few mV. Hyperpolarization to either ACh or CPA was abolished with apamin (50 nM, SKCa blocker) but unaltered by 1-[(2-chlorophenyl) diphenylmethyl]-1H- pyrazole (1 µM TRAM-34, IKCa blocker). During depolarization and contraction to PE, ACh still increased the membrane potential to around -70 mV, but with apamin present the membrane potential only increased just beyond the original resting potential (circa -58 mV). TRAM-34 alone did not affect hyperpolarization to ACh, but in combination with apamin, ACh-evoked hyperpolarization was completely abolished. These data suggest that true endothelium- dependent hyperpolarization of smooth muscle cells in response to ACh is attributable to SKCa, channels, whereas IKCa channels play an important role during the ACh-mediated repolarization phase only observed following depolarization.