To examine mechanisms by which native low-density lipoprotein (n-LDL) perturbs endothelial cell (EC) release of superoxide anion (O₂⁻) and nitric oxide (NO), ECs were incubated with n-LDL at 240 mg cholesterol per deciliter for 4 days with media changes every 24 hours. n-LDL increases EC release of O₂⁻ by more than fourfold and increases nitrite production by 57%. In the conditioned media from day-4 incubations, n-LDL increases total nitrogen oxides 20 times control EC (C-EC) levels. However, n-LDL did not alter EC NO synthase (eNOS) enzyme activity as measured by the [³H]citrulline assay. N^ω-Nitro-l-arginine methyl ester, a specific inhibitor of eNOS activity, increases C-EC release of O₂⁻ by >300% but decreases LDL-treated EC (LDL-EC) release by >95%. l-Arginine inhibits the release of O₂⁻ from LDL-ECs by >95% but did not effect C-EC release of O₂⁻. Indomethacin and SKF 525A partially attenuate LDL-induced increases in O₂⁻ production by ≈50% and 30%, respectively. Thus, n-LDL increases O₂⁻ and NO production, which increases the likelihood of the formation of peroxynitrite (ONOO⁻), a potent oxidant. n-LDL increases the levels of nitrotyrosine, a stable oxidation product of ONOO⁻, and tyrosine by ≈50%. In spite of this increase in oxidative metabolism, analysis of thiobarbituric acid substances reveals that no significant changes in the oxidation of n-LDL occur during the 24-hour incubations with ECs. These data indicate that n-LDL directly perturbs endothelial oxidative metabolism and uncouples l-arginine metabolism from NO release to increase eNOS generation of O₂⁻. Such changes may represent one of the earliest EC perturbations in atherogenesis.