We previously reported enhanced expression of the p67^phox and gp91^phox components of NAD(P)H oxidase in angiotensin (Ang) II–induced hypertension, suggesting de novo assembly in response to Ang II. To examine the direct involvement of NAD(P)H oxidases in Ang II–induced O₂⁻ production, we designed a chimeric peptide that inhibits p47^phox association with gp91^phox in NAD(P)H oxidase (gp91ds-tat). This was achieved by linking a 9-amino acid peptide (aa) derived from HIV-coat protein (tat) to a 9-aa sequence of gp91^phox (known to interact with p47^phox). As a control, we constructed a chimera containing tat and a scrambled gp91 sequence (scramb-tat). We found that gp91ds-tat decreased O₂⁻ levels in aortic rings treated with Ang II (10 pmol/L) but had no effect on either the O₂⁻-generating enzyme xanthine oxidase or potassium superoxide–generated O₂⁻. We infused vehicle, Ang II (0.75 mg · kg⁻¹ · d⁻¹), Ang II+gp91ds-tat (10 mg · kg⁻¹ · d⁻¹), or Ang II+scramb-tat intraperitoneally in C57Bl/6 mice and measured systolic blood pressure (SBP) on days 0, 3, 5, and 7 of infusion. SBP increased by day 3 in mice given Ang II and Ang II+scramb-tat but was significantly lower with Ang II+gp91-tat. On day 7, SBP was still significantly inhibited in mice given Ang II+gp91ds-tat, whereas Ang II–induced O₂⁻ production was inhibited throughout the aorta as detected by dihydroethidium staining, consistent with the ability of this inhibitor to block the various vascular NAD(P)H oxidase isoforms. These data support the hypothesis that inhibition of the interaction of p47^phox and gp91^phox (or its homologues) can block O₂⁻ production and attenuate blood pressure elevation in mice.