We have previously reported that angiotensin II stimulation increased superoxide anion (O₂^-) through the activation of NAD(P)H oxidase and inhibited nitric oxide (NO)-dependent control of myocardial oxygen consumption (MVO₂) by scavenging NO. Our objective was to investigate the role of NAD(P)H oxidase, especially gp91^phox subunit, in NO-dependent control of MVO₂. MVO₂ in mice with defects in the expression of gp91^phox (gp91^phox (-/-)) was measured with a Clark-type oxygen electrode. Baseline MVO₂ was not significantly different between wild-type and gp91^phox (-/-). Stimulation of NO production by bradykinin induced significant decreases in MVO₂ in wild-type. Bradykinin-induced reduction in MVO₂ was enhanced in gp91phox (-/-). Bradykinin-induced reduction in MVO₂ in wild-type was attenuated by 10^-8 mol/L angiotensin II, which was restored by co-incubation with Tiron or apocynin. In contrast to wild-type, bradykinin-induced reduction in MVO₂ in gp91^phox (-/-) was not altered by angiotensin II. There was a decrease in lucigenin (5x10^-6 mol/L) -detectable O₂^- in gp91^phox (-/-) compared to wild-type. Angiotensin II resulted in significant increases in O₂^- production in wild-type, which was inhibited by co-incubation with Tiron or apocynin. However, angiotensin II had no effect on O₂^- production in gp91^phox (-/-). Histological examination showed the development of abscess and/or invasion of inflammatory cell occurred in lungs and livers, but not heart and kidney from gp91^phox (-/-). These results indicate that gp91^phox subunit of NAD(P)H oxidase mediates O₂^- production through the activation of NAD(P)H oxidase and attenuation of NO-dependent control of MVO₂ by angiotensin II.