Supraphysiological increases in serum triglycerides and cholesterol often occur during pregnancy, but their effects on vascular function are poorly understood. Intraperitoneal injection of the nontoxic surfactant poloxamer 407 (P-407) results in sustained elevation of triglycerides and cholesterol. We asked if P-407-induced hyperlipidemia during late pregnancy adversely affects mesenteric resistance artery vasodilator function. On days 13-15 of pregnancy, rats were given a single intraperitoneal injection of P-407, sterile water vehicle, or non-lipid-altering pluronic F-88 (P-88). Four days postinjection, serum triglycerides, cholesterol, free fatty acids, and the lipid peroxidation product malondialdehyde were significantly increased in P-407-treated rats. Mesenteric arteries from P-407-treated rats displayed significant increases in myogenic reactivity (constrictor responses to step increases in intraluminal pressure). The nitric oxide (NO) blocker N(alpha)-methyl-L-arginine increased the myogenic response in control but not in P-407 arteries, normalizing group differences. Endothelial removal increased myogenic reactivity beyond that of prior NO synthase inhibition in controls and potentiated myogenic reactivity in P-407 arteries such that responses again converged. Relaxation responses to the endothelium-dependent vasodilator methacholine did not differ. We conclude that that P-407-induced hyperlipidemia during pregnancy increases myogenic reactivity due to selective attenuation of an NO-mediated vasodilator component of the myogenic response.