1. The involvement of intracellular 3′:5′-cyclic AMP in the inhibition of platelet aggregation by prostaglandin E1, isoprenaline and adenosine has been examined by a radiochemical technique. Platelet-rich plasma was incubated with radioactive adenine to incorporate 14C radioactivity into platelet nucleotides. Pairs of identically treated samples were taken, one for the photometric measurement of platelet aggregation induced by ADP, the other for estimation of the radioactivity of 3′:5′-cyclic AMP. 2. Theophylline, papaverine, dipyridamole and 2,6-bis-(diethanolamino)-4-piperidinopyrimido[5,4d]pyrimidine (compound RA233) were found to inhibit 3′:5′-cyclic AMP phosphodiesterase from platelets. At concentrations of 3′:5′-cyclic AMP greater than 50μm the most active inhibitor was dipyridamole; at 3′:5′-cyclic AMP concentrations less than 19μm, papaverine and compound RA233 were more active than dipyridamole. 3. In the presence of compound RA233 (50μm), the effectiveness of prostaglandin E1 as an inhibitor of platelet aggregation was increased tenfold. Compound RA233 also increased the stimulation by prostaglandin E1 of the incorporation of radioactivity into 3′:5′-cyclic AMP. 4. Compound RA233 (50μm) increased the effectiveness of both adenosine and 2-chloroadenosine as inhibitors of aggregation by 70–100-fold, and in the presence of compound RA233 both adenosine and 2-chloroadenosine stimulated the incorporation of radioactivity into 3′:5′-cyclic AMP; the extent of the stimulation was proportional to the logarithm of the nucleoside concentration. 5. Compound RA233 (100–500μm) inhibited platelet aggregation by itself and caused small increases in the radioactivity of 3′:5′-cyclic AMP. Partial positive correlations were found between the radioactivity of 3′:5′-cyclic AMP in platelets measured at the time of addition of the aggregating agent (ADP) and the extent to which the aggregation was inhibited. 6. The results are interpreted as indicating that adenosine, 2-chloroadenosine, isoprenaline, prostaglandin E1 and drugs that inhibit platelet 3′:5′-cyclic AMP phosphodiesterase all inhibit aggregation by a common mechanism involving intracellular 3′:5′-cyclic AMP.