Although high-density lipoprotein (HDL) has been found to decrease platelet function per se, little is known regarding the mechanism of its platelet inhibitory effect. In this study, we confirmed the inhibitory effect of HDL on platelet aggregation and 14C-serotonin release in thrombin-activated washed human platelets. The inhibition of platelet function was associated with an increase in nitric oxide synthase activity, measured as the conversion of 3H-L-arginine to 3H-L-citrulline as well as nitrite release in the platelet supernates. The inhibition of platelet function by HDL was reversed by preincubation of washed platelets with an inhibitor of nitric oxide synthase, NG-nitro-L-arginine methyl ester (L-NAME), and potentiated by co-incubation with the precursor of nitric oxide, L-arginine. These observations suggest that HDL decreases platelet function by increasing nitric oxide synthase activity in human platelets.