We have characterized the NO synthase enzyme in the villous vasculature of the human placenta as part of our ongoing studies of the regulation of NO synthesis in this circulation. NO synthase activity was determined by conversion of 3H l-arginine to 3H l-citrulline in cellular homogenate, cytosolic and particulate fractions. Optimal NO synthase activity was measured in all fractions in the presence of 1 mm NADPH, 10 μm tetrahydrobiopterin, 2 μm FAD, 100 μm free calcium and 50 U/ml calmodulin. The calmodulin inhibitor calmidazolium (50 μm) and FAD inhibitor diphenyliodonium chloride (1 μm) significantly reduced enzyme activity. The EC50 for calcium was 0.1 μm and Km for l-arginine 2.00±0.49 μm with Vmax 55.8±28.3 pmoles/mg protein/min. Enzyme activity was inhibited in both cytosolic and particulate fractions by ng-nitro-l-arginine and ng-monomethyl-l-arginine in a concentration-dependent manner (10−8–10−4 m). A calcium-independent NO synthase activity was also determined, but only constituted between 5–6 per cent of total activity. On Western blotting, a single 135 kda species was identified in each fraction with a monoclonal antibody raised against bovine aortic endothelial NO synthase. The NO synthase enzyme of the villous vasculature appears to correspond to the type III calcium-calmodulin dependent endothelial isoform. © 1993, Baillière Tindall. All rights reserved.