We hypothesized that 3',4'-dihydroxyflavonol (DiOHF) by scavenging superoxide anions (O2-*) would increase the bioavailability of NO and potentiate NO-mediated relaxation in the rat aorta. Furthermore we hypothesized that DiOHF, by its antioxidant activity, would preserve responses to acetylcholine (ACh) in the presence of O2-* generators in the aorta in vitro and after ischemia and reperfusion of the rat hindquarters vasculature in situ. Using lucigenin-enhanced chemiluminescence we demonstrated that DiOHF caused a concentration-dependent reduction in O2-* accumulation whether generated by xanthine/xanthine oxidase in a cell-free system or by rat isolated aorta in the presence of NADPH. DiOHF also prevented the inhibitory effects of xanthine/xanthine oxidase and pyrogallol on vasorelaxation to ACh and sodium nitroprusside (SNP) in the rat aorta in vitro, and attenuated the vascular dysfunction caused by 2 h ischemia and 2 h reperfusion (I/R) in the rat hindquarters. I/R significantly reduced the dilator responses to both ACh and SNP; however, this effect was attenuated when DiOHF was given before the onset of ischemia or reperfusion. In conclusion, DiOHF, by scavenging O2-*, increases the relaxant activity of ACh and SNP and reduces the degree of inhibition of xanthine/xanthine oxidase or pyrogallol on the response to ACh. DiOHF reduces the adverse effects of I/R on vascular function by increasing NO bioavailability suggesting that it may be useful in preventing reperfusion injury.