In vascular tissues, an NAD(P)H oxidase is the main source of superoxide; however, there has been much uncertainty regarding its activity and the levels of superoxide it generates. This problem has limited overall progress in this field. Therefore, studies were performed and techniques developed to quantitatively assess the function of the vascular NAD(P)H oxidase, measuring its rate of superoxide production and substrate consumption in rat aortic homogenates and intact segments. NADPH/NADH oxidation was measured spectrophotometrically, and oxygen consumption was measured by electrochemical probe. Superoxide was detected and quantitated by electron paramagnetic resonance spin trapping. Under basal conditions, superoxide generation and oxygen consumption were negligible. After addition of NADPH or NADH (0.1 mM), superoxide was generated at rates of 0.41 +/- 0.03 or 0.36 +/- 0.04 nmol x mg protein(-1) x min(-1), respectively. Oxygen was consumed with a similar time course at rates of 1.5 +/- 0.2 or 1.3 +/- 0.3 nmol. mg protein(-1) x min(-1), and NADPH or NADH were oxidized at rates of 1.8 +/- 0.4 and 1.5 +/- 0.3 nmol x mg protein(-1) x min(-1), respectively. In intact aortic rings, superoxide was generated with rates of 4.0 +/- 0.7 or 3.7 +/- 0.7 pmol x mg tissue(-1) x min(-1), whereas oxygen was consumed at rates of 22.1 +/- 5.0 or 14.5 +/- 3.3 pmol x mg tissue(-1) x min(-1), for NADPH or NADH, respectively. These values are lower than those previously measured using lucigenin, which uncouples flavoenzymes, triggering additional superoxide generation. This quantitative approach for characterization of the vascular NAD(P)H oxidase activity should facilitate the further identification and cellular characterization of this enzyme(s) and its functional and signaling roles.