The relation of cyclic 3',5'-guanosine monophosphate (cGMP) to platelet function was studied by investigating the influence of this compound and its N2,O2'-dibutyryl derivative (DBcGMP) on platelet aggregation and the release reaction. Both cGMP and dibutyryl cGMP enhanced to an equal extent platelet aggregation induced by epinephrine or by chick skin collagen alpha1 chain. The platelet release reaction, as measured by the release of [14C]-labeled 5-hydroxytryptamine (serotonin) was also enhanced by the cyclic nucleotides. Both compounds were also able to partially overcome the inhibitory effect of N6,O2'-dibutyryl 3',5'-adenosine monophosphate (DBcAMP), prostaglandin E1, and theophylline. The effect of DBcGMP did not result from contamination with 5'-GMP. Butyric acid and 5'-GMP, either alone or in combination, had no detectable effect on platelet function. The in vitro effect of DBcGMP and cGMP was not dependent on preincubation of platelets with the compounds. This suggests that their effect is mediated by direct action on the platelet membranes. These data are consistent with previous observations that the platelet aggregating agents (epinephrine, ADP, collagen, and chick collagen alpha1 chains) cause an increase in the content of GMP, and support the hypothesis that platelet aggregation is favored by an increase in cGMP.