We studied the regulation of nitric oxide (NO) synthase activity in the cytosol from freshly isolated (native) as well as cultured porcine aortic endothelial cells (PAECs). l-Arginine-dependent NO synthesis was detected by activation of a purified soluble guanylyl cyclase (GC) incubated with cytosol in the presence of nicotinamide adenine dinucleotide phosphate (NADPH). In cytosol from native PAECs, NO synthesis was increased by Ca2+ ions in a concentration- and calmodulin-dependent fashion. This increase was inhibited by calmodulin-binding proteins (melittin, calcineurin) and by removal of endogenous calmodulin by anion-exchange chromatography. The inhibition was overcome by addition of calmodulin from porcine brain and from heat-denatured cytosol. In cytosol from cultured PAECs, specific NO synthase activity was at least threefold lower than in native cells, and was about twofold enhanced by calcium/calmodulin. In the presence of maximally effective concentrations of l-arginine (0.3 mM), NADPH (0.1 mM), free calcium ions (2 μM), and calmodulin (1 μM), (6R)-tetrahydrobiopterin (0.1 μM) increased NO synthase activity about 1.5-fold in native and threefold in cultured endothelial cells. NG-nitro-l-arginine (0.3 mM) completely inhibited Ca2+/calmodulin- and tetrahydrobiopterin-dependent NO synthase. Other biopterins, pteridins, and tetrahydrofolic acid were ineffective. We conclude that calmodulin links agonist-induced calcium transients in the endothelium with NO formation. Tetrahydrobiopterin is required as a cofactor for expression of maximal NO synthase activity in isolated endothelial cytosol.