Nitric oxide is a recently discovered biomolecule with a broad range of actions and synthesized by nitric oxide synthases. We investigated effects of lipids on particulate nitric oxide synthase purified from cultured bovine aortic endothelial cells by monitoring the conversion of L-[3H]arginine to L-[3H]citrulline. Phosphatidylcholine, lysophosphatidylcholine, and phosphatidylethanolamine dose-dependently enhanced the enzyme activity up to 3 fold in the presence of Ca2+, calmodulin, NADPH, FAD, and (6R)-5,6,7,8-tetrahydrobiopterin. These phospholipids increased the Vmax of nitric oxide synthase without altering the Km for L-arginine and the affinities for Ca2+ and calmodulin. These findings suggest that phospholipids play an important role in modulating endothelial nitric oxide synthase activity.