N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES) is a commonly-used buffer. This study determined the effect of HEPES on contractility of the dog basilar artery and tested the hypothesis that HEPES inhibits vasoconstriction of isolated arterial segments by generating H2O2. Rings of dog basilar artery with or without endothelium were suspended under isometric tension and contracted with KCl, serotonin, or prostaglandin F2 alpha (PGF2 alpha) in bicarbonate or HEPES buffer. Addition of HEPES, 30 mmol l-1, before or after contraction with KCl, serotonin or PGF2 alpha significantly decreased maximal tension in rings with or without endothelium. Preincubation with HEPES buffer, 10 mmol l-1, significantly decreased maximal contractions to each agonist in rings with endothelium and to KCl and serotonin in rings without endothelium. HEPES, 30 mmol l-1, noncompetitively inhibited concentration-contraction curves to increasing concentrations of each agonist in rings with or without endothelium. Inhibition by HEPES was completely reversible with washing. The inhibitory effects of HEPES on responses to each agonist in rings with endothelium were significantly less in the dark or after coincubation with catalase. Unlike HEPES, effects of H2O2 were endothelium-dependent in that H2O2 caused contractions in rings with endothelium and relaxations in rings without endothelium. 5-(N,N'-dimethyl)-amiloride and 4,4'-diisothiocyanataostilbene-2,2'-disulfonic acid did not affect contractility in this preparation. These results show that HEPES exerts significant inhibitory effects on arterial smooth muscle contractility. The mechanism does not involve endothelium-dependent relaxation, effects on chloride channels or the sodium-hydrogen exchanger or generation of H2O2 by HEPES in the light.