We previously reported increased aortic reactive oxygen species (ROS) production in mineralocorticoid (deoxycorticosterone acetate [DOCA]-salt) hypertensive rats. In the present study, we tested the hypothesis that NADH/NADPH oxidase is responsible for increased ROS production, namely superoxide (O₂⁻), in aorta from the DOCA-salt rat. Treatment of aortic rings from DOCA-salt rats with the NO synthase inhibitor N-nitro-l-arginine and the xanthine oxidase inhibitor allopurinol did not significantly change O₂⁻ production. Furthermore, de-endothelialization of aorta from DOCA-salt rats did not affect O₂⁻ production compared with that of sham-operated rats. Thus, xanthine oxidase and uncoupled endothelial NO synthase were not responsible for increased O₂⁻ production in the DOCA-salt rats. In contrast, treatment with the NADPH oxidase inhibitor apocynin significantly decreased O₂⁻ production in aortic rings from DOCA-salt rats compared with sham-operated rats. Moreover, long-term administration of apocynin (in drinking water, 1.5 mmol/L, 28 days) to DOCA-salt rats significantly decreased systolic blood pressure compared with that of rats treated with DOCA-salt alone. Furthermore, O₂⁻ production in aortic rings from DOCA-salt rats treated with apocynin for 28 days was reduced compared with that of untreated DOCA-salt rats. Reverse transcriptase–polymerase chain reaction (RT-PCR) analysis demonstrated that DOCA-salt rats have significantly greater mRNA levels of the NADPH oxidase subunit p22phox than do sham-operated rats. These findings suggest that NADPH oxidase is increased and is responsible for increased O₂⁻ production and possibly contributes to increased blood pressure in the DOCA-salt hypertensive rat.