HIV-associated cardiovascular diseases have been widely described, but clinical studies aimed at establishing cause-effect relationships between HIV-associated cardiovascular disease and either the HIV infection or antiretroviral therapy have been problematic. Endothelial dysfunction is a sensitive marker and early event in atherosclerosis, and many have suggested that protease inhibitors promote endothelial dysfunction indirectly by inducing elevations in circulating lipids. To determine whether nucleoside reverse transcriptase inhibitors and/or protease inhibitors induce endothelial dysfunction, and to test whether this effect is dependent upon drug-mediated alteration in plasma lipid concentrations, we treated male Sprague-Dawley rats with pharmacological doses of azidothymidine (AZT), indinavir, or AZT plus indinavir through their drinking water for 1 month and assessed endothelial function in aortic rings using an isometric force measurement. Circulating levels of plasma lipids and endothelin-1, a marker for endothelial injury and/or dysfunction, were also determined. We found that AZT and AZT plus indinavir treatments dramatically reduced endothelium-dependent vessel relaxation. However, AZT treatment did not significantly alter plasma levels of cholesterol or triglyceride. In addition, plasma endothelin-1 levels were elevated in rats treated with AZT plus indinavir. Indinavir treatment alone increased plasma cholesterol levels but had no effect on endothelial function. These findings suggest that in addition to modulating plasma lipid levels, antiretrovirals, particularly AZT and perhaps other nucleoside reverse transcriptase inhibitors, may have direct effects on the vascular endothelium. Together with other increased risk factors for atherosclerosis in HIV patients, AZT-induced endothelial dysfunction may contribute to the cardiovascular diseases associated with HIV antiretroviral therapy.