NADPH-oxidase is now regarded as the major source of reactive oxygen species (ROS) in the vasculature, and sustained high levels of ROS such as superoxide (O2-) are known to exert damaging, pro-constrictor effects in blood vessels. However, ROS can also act as endogenous signalling molecules within some blood vessels, especially when generated acutely in low levels. In particular, hydrogen peroxide (H2O2) generated following activation of NADPH-oxidase is an effective cerebral vasodilator in vivo, raising the possibility that this enzyme could play a physiological role, at least in the cerebral circulation. However, it is unknown whether NADPH-oxidase activity and function varies between vascular beds. Hence, the first aim of this study was to compare NADPH-oxidase activity in arteries from the rat intracranial (basilar, BA; middle cerebral, MCA) and systemic (carotid, CA; mesenteric, MA; renal, RA; aorta, AO) circulations of Sprague-Dawley rats. Furthermore, although the activity of NADPH-oxidase is reportedly suppressed in the systemic circulation of females, it is unknown whether such gender differences exist in cerebral blood vessels. Therefore, a second aim was to test whether female gender is associated with lower NADPH-oxidase activity and function in the cerebral circulation. NADPH (100 mol/L)-stimulated O2- production was measured by lucigenin-chemiluminescence. Additionally, ring segments of BA, AO, CA and MA were mounted in a wire myograph or organ bath for recording of isometric tension. NADPH-stimulated O2- production by BA (5312431) and MCA (113361020) was 10-80-fold greater than levels generated by AO (53440), CA (14018), MA (23842) and RA (28732) (P MA>AO=CA. Endothelial removal inhibited NADPH-induced relaxation of BA by >50%, whereas it increased relaxation of AO, CA and MA in response to NADPH. Treatment with the H2O2 scavenger catalase (1000 units/ml) also prevented NADPH-induced relaxation of BA. Finally. both relaxation of BA and production of O2- in response to NADPH was 50% smaller in females (P<0.05), whereas relaxation responses to H2O2 were similar between genders. Studies in ovariectomized female rats treated with either vehicle or 17-estradiol (10 g/kg per d, 14 d) revealed that the gender difference in NADPH-oxidase activity was estrogen-dependent. Thus, NADPH-oxidase activity is profoundly higher in intracranial versus systemic arteries of the rat. Intracranial arteries are also more sensitive to the vasodilator effects of endogenous or exogenous H2O2, consistent with the possibility that ROS derived from NADPH-oxidase play a physiological role as vasodilators in the cerebral circulation. NADPH-oxidase activity and function is suppressed in the female cerebral circulation due to an effect of estrogen.