1. We have identified a neuronal nitric oxide synthase (NOS)-like constitutive form of NOS in vascular smooth muscle (VSM) using a functional contractility approach as well as immunohistochemical methods. 2. N(G)-Nitro-L-arginine methyl ester, N(G)-monomethyl-L- arginine and N(G)-nitro-L-arginine (L-NOARG), the competitive inhibitors of NOS, inhibited Mg(2+)-induced relaxation of de-endothelialized rat aorta precontracted with phenylephrine (PE). This Mg(2+) relaxation of VSM was not affected by inhibitors of inducible NOS. 3. Electrical field stimulation (EFS; 30-70 Hz) caused relaxation of rat aorta in the presence of tetrodotoxin (therefore not a neurogenic effect) and this EFS relaxation was effectively inhibited by L-NOARG, oxyhemoglobin and methylene blue. 4. Immunohistochemical studies of dog saphenous vein using antibodies raised against neuronal NOS indicated prominent staining along the plasmalemma in a punctate pattern similar to the distribution of antibodies against caveolin-1, a major constituent of the plasmalemmal caveolae. 5. We propose that a constitutive NOS of non-endothelial, non-neuronal origin is present in a special caveolae domain of VSM cell membranes and could be activated by an ionic mechanism yet to be characterized.