1. The activation of the L-arginine:nitric oxide (NO) pathway during aggregation of human platelets by adenosine 5'-diphosphate (ADP), arachidonic acid, thrombin and the calcium ionophore A23187 and its inhibition by N(G)-monomethyl-L-arginine (L-NMMA), N(G)-nitro-L-arginine methyl ester (L-NAME) and N-iminoethyl-L-ornithine (L-NIO) were studied. The inhibition of the cytosolic platelet NO synthase by these compounds was also examined. 2. Platelet aggregation induced by ADP (1-10 μM) and arachidonic acid (0.1-10 μM), but not that induced by thrombin (1-30 mu ml-1) or A23187 (1-10 nM), was inhibited by L-, but not D-arginine (1-30 μM). However, in the presence of a subthreshold concentration of prostacyclin (0.1 nM) or of M and B 22948 (1 μM), a selective inhibitor of guanosine 3':5'-cyclic monophosphate (cyclic GMP) phosphodiesterase, L-arginine caused concentration-dependent inhibition of aggregation induced by all of these aggregating agents. 3. L-NMMA, L-NAME and L-NIO (all at 1-30 μM), but not their D-enantiomers, enhanced to the same extent platelet aggregation induced by ADP, arachidonic acid and thrombin without affecting that induced by A23187. 4. In the presence of 300 μM L-arginine, the NO synthase in platelet cytosol was inhibited by L-NMMA, L-NAME and L-NIO with IC50s of 74 ± 9, 79 ± 8 and 8.5 ± 1.5 μM (n = 3), respectively. 5. These results indicate that the L-arginine:NO pathway in human platelets play a role in the modulation of platelet aggregation.