ADP mediates platelet-induced relaxation of blood vessels and may function as an important intercellular signaling molecule in the brain. We used pharmacological and genetic approaches to examine mechanisms that mediate responses of cerebral arterioles to ADP, including the role of endothelial nitric oxide synthase (eNOS). We examined responses of cerebral arterioles (control diameter approximately 30 microm) in anesthetized wild-type (WT, eNOS+/+) and eNOS-deficient (eNOS-/-) mice using a cranial window. In WT mice, local application of ADP produced vasodilation that was not altered by indomethacin but was reduced by approximately 50% by NG-nitro-L-arginine (L-NNA) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (inhibitors of NOS and soluble guanylate cyclase, respectively). In eNOS-/- mice, responses to ADP were largely preserved, and a significant component of the response was resistant to L-NNA (a finding similar to that in WT mice treated with L-NNA). In the absence of L-NNA, responses to ADP were markedly reduced by charybdotoxin plus apamin [inhibitors of Ca2+-dependent K+ channels and responses mediated by endothelium-derived hyperpolarizing factor (EDHF)] in both WT and eNOS-/- mice. Thus pharmacological and genetic evidence suggests that a significant portion of the response to ADP in cerebral microvessels is mediated by a mechanism independent of eNOS. The eNOS-independent mechanism is functional in the absence of inhibited eNOS and most likely is mediated by an EDHF.