Our goals were to determine whether chronic exposure to nicotine alters nitric oxide synthase (NOS)-dependent reactivity of cerebral (pial) arterioles and to identify a potential role for NAD(P)H oxidase in impaired NOS-dependent responses during chronic exposure to nicotine. We measured in vivo diameter of pial arterioles to NOS-dependent (acetylcholine and 5'-adenosine diphosphate (ADP)) and -independent (nitroglycerin) agonists in saline treated rats and rats chronically treated with nicotine (2mg/kg/day for 2 weeks via an osmotic minipump). We found that NOS-dependent, but not -independent, vasodilatation was impaired in nicotine treated compared to saline treated rats. In addition, the production of superoxide anion (lucigenin chemiluminescence) was increased in rats treated with nicotine when compared to saline treated rats. Further, using Western blot analysis, we found that chronic exposure to nicotine increased p47phox protein in the parietal cortex. Finally, we found that apocynin (40 mg/kg/day in the drinking water to inhibit NAD(P)H oxidase alleviated impaired NOSdependent cerebral vasodilatation in nicotine treated rats, did not alter NOS-dependent responses in saline treated rats, and did not alter NOS-independent reactivity in saline or nicotine treated rats. These findings suggest that chronic exposure to nicotine impairs NOS-dependent dilatation of pial arterioles by a mechanism that appears to be related to the formation of superoxide anion via activation of NAD(P)H oxidase.