We previously reported that NADPH oxidase activity is greater in intracranial cerebral versus systemic arteries of the rat. Here, we first tested whether NADPH oxidase activity is also greater in intracranial cerebral than systemic arteries of three other animal species, i.e., mouse, rabbit, and pig. Second, using Nox2-deficient mice, we evaluated the involvement of Nox2-containing NADPH oxidases in any such regional differences. NADPH-stimulated superoxide (O2−) production by basilar, middle cerebral arteries (MCA), and common carotid arteries (CA) and thoracic aorta (AO) from rat, mouse, rabbit, and pig was measured using lucigenin-enhanced chemiluminescence. Basal production of O2− and hydrogen peroxide (H2O2) by cerebral arteries, AO, and CA from wild-type (WT) and Nox2−/− mice was measured using L-012-enhanced chemiluminescence and Amplex Red fluorescence, respectively. Western blotting was used to measure Nox2 and SOD1–3 protein expression, and immunofluorescence was used to localize Nox2, in mouse arteries. In rats, WT mice, rabbits, and pigs, NADPH-stimulated O2− production by cerebral arteries was up to 40-fold greater than that in AO and CA. In WT mice, basal O2− and H2O2 production by cerebral arteries was ninefold and ∼2.5-fold higher, respectively, than that in AO and CA and was associated with ∼40% greater expression of Nox2 protein. Nox2 immunofluorescence was localized to the endothelium, and to a lesser extent the adventitia, in all mouse arteries and appeared to be more intense in endothelium of MCA than AO or CA. In Nox2−/− mice, NADPH-stimulated O2− production by cerebral arteries was ∼35% lower than that in WT mice, whereas Nox2 deletion had no significant effect on O2− production by AO or CA. Thus NADPH oxidase activity is greater in intracranial cerebral versus systemic arteries of several animal species and is associated with higher cerebrovascular expression and activity of Nox2.