Although the endothelium co-generates both NO and endothelium derived hyperpolarising factor (EDHF), the relative contribution from each vasodilator is not clear. In studies where the endothelium is stimulated acutely, EDHF responses predominate in small arteries. However, the temporal relationship between endothelial derived NO and EDHF over more prolonged periods is unclear, but of major physiological importance. Here we have used a classical pharmacological approach to show that EDHF is released transiently compared with NO. Acetylcholine (3x10^-6mol/L) dilated second and/or third order mesenteric arteries for prolonged periods of up to 1 hour, an effect that was reversed fully and immediately by the subsequent addition of L-NAME, (10^-3mol/L), but not TRAM-34 (10^-6mol/L) plus apamin (5x10^-7mol/L). When vessels were pre-treated with L-NAME acetylcholine induced relatively transient dilator responses (declining over approximately 5 min), and sensitive to TRAM-34 plus apamin. When measured in parallel, the dilator effects of acetylcholine outlasted the smooth muscle hyperpolarisation. However, in the presence of L-NAME vasodilatation and hyperpolarisation followed an identical time course. In vessels from NOSIII^-/-, mice acetylcholine induced small, but detectable dilator responses, which were transient in duration and blocked by TRAM-34 plus apamin. EDHF responses in these mouse arteries were inhibited by an intracellular calcium blocker, TMB-8, and the phospholipase A₂ inhibitor, AACOCF₃, suggesting a role for lipid metabolites. These data show for the first time that EDHF is released transiently whilst endothelial derived NO is released in a sustained manner.