The goal of this study was to determine the role of endothelium-derived relaxing factor (EDRF) in dilatation of cerebral arterioles in response to adenosine 5’-diphosphate (ADP). Using intravital microscopy, we measured the diameters of pial arterioles in vivo in rats during superfusion with ADP, acetylcholine and nitroglycerin before and during topical application of inhibitors of nitric oxide synthesis (N<sup>G</sup>-monomethyl- L-arginine; L-NMMA and N<sup>w</sup>-nitro-L-arginine; L-NNA). Prior to suffusion with L-NMMA and L-NNA, ADP (10 and 100 µ M) , acetylcholine (0.1 and 1.0 µ M) , and nitroglycerin (1.0 and 10 µ M)produced dose-related increases in the diameter of pial arterioles. Following application of L-NMMA (1.0 and 10 µ M)and L-NNA (0.1 and 1.0 µ M) , dilatation of cerebral arterioles in response to ADP was significantly inhibited. In addition, L-NMMA and L-NNA significantly inhibited dilatation of cerebral arterioles in response to acetylcholine, but did not alter vasodilatation in response to nitroglycerin. Thus, our findings suggest that ADP dilates cerebral arterioles via the production of nitric oxide, or a nitric-oxide-containing compound.