When blood flow in a large artery is increased the vessel dilates. This flow-dependent dilation requires endothelial cells, and is not mediated by an ascending message from the microcirculation or a myogenic mechanism. Adrenergic, cholinergic, or ganglionic blockade does not alter the dilation response. Inhibition of cyclo-oxygenase by indomethacin has no effect, but inhibition of both lipoxygenase and cyclo-oxygenase by 5,8,11,14-eicosatetraynoic acid (ETYA) inhibits the dilation and shifts the acetylcholine dose response curve to the right. Inhibition of guanylate cyclase by methylene blue blocks the dilation response and shifts the acetylcholine dose response curve to the right. This suggests that both cyclic GMP and a nonprostaglandin metabolite of arachidonic acid are involved in the dilation response to increased flow. We propose that increased blood flow initiates an initial response, which results in endothelial cell production and release of a nonprostaglandin metabolite of arachidonic acid. This metabolite stimulates vascular smooth muscle guanylate cyclase, leading to increased cyclic GMP and vasodilation.