To investigate whether insulin reduces platelet aggregability through a modulation of the guanosine-3′,5′-cyclic monophosphate (cGMP) concentrations, we determined by a radioimmunoassay the cGMP values in the platelet-rich plasma (PRP) obtained from 17 healthy volunteers and incubated for 3 min with different concentrations of human recombinant insulin (0, 240, 480, 720, 960, and 1,920 pM). Insulin induced a dose-dependent cGMP increase, from 18.5 ± 3.3 to 42.0 ± 6.4 pmol/10⁹ platelets (P = 0.0001). This increase was completely blunted when PRP was preincubated for 20 min with the tyrosine kinase inhibitor genistein (10 μM) or with the guanylate cyclase inhibitor methylene blue (10 μM), but the increase remained highly significant (P = 0.003 and 0.009) when PRP was preincubated for 20 min with the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX, 500 μM) or with the nitric oxide synthase inhibitor N^G-mono-methyl-L-arginine (L-NMMA, 30 μM). Finally, the insulin-induced decrease of platelet aggregability to collagen and ADP was completely blunted when PRP was preincubated with 10 μM of the guanylate cyclase inhibitor methylene blue. This study demonstrates that the platelet anti-aggregatory effect exerted by insulin is attributable to the insulin-induced increase of cGMP that is due to a direct receptor-mediated platelet guanylate cyclase activation.