Our goals were to determine whether acute exposure to nicotine alters neuronal nitric oxide synthase (nNOS) dependent reactivity of cerebral arterioles and to identify a potential role for oxidative stress in nicotine induced impairment in nNOS dependent responses of cerebral arterioles. We measured in vivo diameter of cerebral arterioles to nNOS dependent (NMDA and kainate) and independent (nitroglycerin) agonists before and during acute treatment with nicotine. We found that nNOS dependent, but not independent, vasodilatation was impaired during treatment with nicotine. In addition, treatment of the cerebral microcirculation with tempol (1 hour prior to infusion of nicotine) prevented nicotine induced impairment in nNOS dependent vasodilatation. Further, the production of superoxide anion (lucigenin chemiluminescence) was increased in parietal cortex tissue of rats by treatment with nicotine and this increase in superoxide anion production could be inhibited by tempol. Our findings suggest that acute exposure to nicotine impairs nNOS dependent dilatation of cerebral arterioles by a mechanism that appears to be related to the formation of superoxide anion.