It is unclear whether the abnormal relaxation seen in diabetes is due to decreased levels of nitric oxide (NO) and how eicosapentaenoic acid (EPA, C20:5 omega 3) affects the endothelial production of NO. We investigated the effects of EPA ethyl ester (EPA-E) and elevated glucose on NO production by human endothelial cells (HUE). EPA-E (0.3 mM) significantly enhanced [NO2] production and the intracellular concentration of free Ca2+ within 3 min after EPA-E was added to the cultures. High levels of glucose (27.5 mM) significantly increased endothelial glucose, sorbitol and fructose, and inhibited [NO2-] production. However, EPA-E (0.3 mM) prevented the inhibition of [NO2-] production due to the activation of the Ca(2+)-calmodulin system of NO synthase. EPA-E decreased the glucose-mediated inhibition of NO production by HUE. These results suggest this agent might ameliorate endothelial dysfunction associated with diabetes.