Nitric oxide originating from the endothelial cells of the vessel wall is essential for the vascular system. It is produced by the enzyme endothelial nitric oxide synthase (eNOS). Cellular eNOS activity is affected by changes in eNOS synthesis. To address whether degradation also contributes to eNOS activity, the effect of proteasome inhibitors on eNOS-mediated NO synthesis was studied in the microvascular endothelial cell line bEnd.3 and in cultured primary aortic endothelial cells. Surprisingly, agonist-induced increases in eNOS activity were reduced to 42 and 50% in the presence of the proteasome inhibiting drugs MG132 and clasto-lactacystin-beta-lactone, respectively (P < 0.01). The decrease in activity occurred within 1 hour of drug treatment and was not accompanied by a change in intracellular levels of either eNOS or its inhibitor caveolin-1. Taken together, these data may indicate that eNOS is regulated by an interacting protein, different from caveolin-1, that inhibits its activity and is rapidly degraded by the proteasome in the presence of eNOS agonists.