Neuropeptide Y (NPY) is an important vasoconstrictor in the cerebral circulation. Its constrictor response is because of activation of NPY receptors on the vascular smooth muscle (VSM). Little is known regarding the effects of NPY on the endothelium. In the current study, the authors tested the hypothesis that NPY can either constrict or dilate rat middle cerebral arteries (MCAs). Constriction is elicited by stimulating receptors on the VSM; dilation is elicited by stimulating receptors on the endothelium. Middle cerebral arteries were isolated, cannulated with micropipettes, pressurized to 85 mm Hg, and luminally perfused. The extraluminal application of NPY (mixed agonist), [Leu³¹, Pro³⁴]-NPY (Y1 agonist), or NPY-[13–36] (Y2 agonist) produced concentration-dependent constrictions. BIBP 3226 (Y1 selective antagonist) significantly attenuated the NPY-and [Leu³¹, Pro³⁴]-NPY–induced constrictions. The luminal application of NPY, [Leu³¹, Pro³⁴]-NPY, and NPY-[13–36] produced concentration-dependent dilations of MCAs. The maximum dilation produced by the NPY receptor agonists was approximately 40% of the dilation elicited by the luminal administration of 10⁻⁵ mol/L ATP. Dilations elicited by luminal NPY, [Leu³¹, Pro³⁴]-NPY, or NPY-[13–36] were abolished by inhibition of nitric oxide synthase with 10⁻⁵ mol/L N^ω-nitro-L-arginine methyl ester (L-NAME) or removal of the endothelium. Dilations produced by luminal NPY or luminal [Leu³¹, Pro³⁴]-NPY were not affected by BIBP 3226. Stimulation of NPY receptors on vascular smooth muscle constricted MCAs. Stimulation of an NPY receptor other than the Y1 subtype on endothelium dilated the MCAs by releasing nitric oxide.