Retrospective epidemiological studies have suggested that antioxidant therapy may decrease cardiovascular morbidity and mortality rates, although the mechanisms for this effect remain unclear. In the present study, we demonstrate that selective antioxidants can enhance expression of endothelial nitric oxide synthase (eNOS). We found that the antioxidants nordihydroguaiaretic acid (NDGA), catechol, glutaryl probucol, and N-acetylcysteine increased eNOS expression in cultured bovine aortic endothelial cells (BAECs). NDGA seemed to be the most potent of the phenolic antioxidants, producing a 3-fold increase in eNOS mRNA. This effect of NDGA was enhanced by nonphenolic antioxidants such as N-acetylcysteine and ascorbic acid. Nuclear run-on studies indicated that NDGA increased eNOS transcription. A similar increase in eNOS protein content was observed with Western blot analysis after treating BAECs or human aortic endothelial cells with NDGA. Exposure of BAECs to NDGA enhanced NO production, as measured by electron paramagnetic resonance spin trapping and eNOS activity, as measured by [14C]arginine-to-[14C]citrulline assay. Methylation of the phenolic hydroxyl groups completely inhibited the NDGA effect on eNOS mRNA levels. This effect of NDGA was not due to inhibition of lipoxygenase because cis-5,8,11,14-eicosatetraynoic acid did not alter eNOS expression. We conclude that antioxidants may not only increase the bioactivity of nitric oxide but also enhance expression of the eNOS enzyme. Such an effect may prove useful in conditions such as hypertension and atherosclerosis, in which nitric oxide production and/or biological activity is impaired.