Arteriolar dilation to endothelium-derived relaxing factor (EDRF) is suppressed early in diabetes mellitus. The purpose of this study was to determine whether acute exposure to a hyperglycemic media can suppress EDRF function of normal arterioles. Dilation of intestinal arterioles to iontophoretically applied acetylcholine (ACh) and nitroprusside was measured in normoglycemic rats before and after 1 h of topical exposure to isotonic solutions containing D-glucose concentrations of 200, 300, and 500 mg/100 ml. Exposure to a D-glucose concentration of 200 mg/100 ml had no effect on vasodilation to ACh. D-Glucose concentrations of both 300 and 500 mg/100 ml caused significant suppression of the responses: for example, at the approximate 50% effective dosage (100 nA), the dilatory response was decreased by 60% at a D-glucose concentration of 300 mg/100 ml and 55% at a D-glucose concentration of 500 mg/100 ml. Responses to nitroprusside were not significantly (P < 0.05) impaired after exposure to D-glucose concentrations of 200, 300, or 500 mg/100 ml. Exposure to an isotonic L-glucose concentration of 500 mg/100 ml for 1 h had no significant (P > 0.05) effect on responses to ACh. Pretreatment with superoxide dismutase, catalase, indomethacin, or meclofenamic acid preserved EDRF-mediated vasodilation during exposure to a D-glucose concentration of 500 mg/100 ml at almost all the ACh dosages tested. These results indicate that oxygen radicals formed in part by increased eicosanoid synthesis during exposure to D-glucose hyperglycemia interfere with the EDRF mechanism before its action on the microvascular smooth muscle.