Purified particulate endothelial nitric oxide (NO) synthase requires NADPH, Ca2+, calmodulin, and 5,6,7,8-tetrahydrobiopterin (BH4) for enzymatic conversion of l-arginine to l-citrulline and NO. We now report that the purified particulate endothelial enzyme has both FAD and flavin mononucleotide bound to the enzyme in equimolar amounts and this amount of bound flavin is sufficient for full activity of the enzyme. Also, we found that over 90% of the biopterin bound to the enzyme is in the reduced form of 5,6,7,8-tetrahydrobiopterin. However, the small amount of bound biopterin is not sufficient for maximal activity.