We previously reported that NADPH oxidase activity is greater in intracranial cerebral versus systemic arteries of the rat. Here, we firstly tested whether NADPH oxidase activity is also greater in intracranial cerebral arteries than systemic arteries of three other animal species, i.e. mouse, rabbit and pig. Secondly, 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 (BA), middle cerebral (MCA) and common carotid (CA) arteries, 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 in AO and CA. In WT mice, basal O₂^- and H₂O₂ production by cerebral arteries was 9-fold and ~2.5-fold higher, respectively, than 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 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.