Purified human blood platelet membrane showed the presence of one low Km (1.1 microM) and one high Km (5.0 microM) cyclic AMP phosphodiesterase(s). Incubation of platelet-rich plasma or gel-filtered platelets with ADP (4.0 microM), a well-known platelet aggregating agent, resulted in the inhibition of phosphodiesterase activity of the isolated membrane by 25% in 5 min at 23 degrees C. A Lineweaver-Burk plot of the enzymic activity of the membrane preparation showed that ADP specifically inhibited the low Km (1.1 microM) phosphodiesterase by reducing the Vmax from 241 to 176 pmol/mg per min with concomitant lowering of Km to 0.5 microM. In contrast, neither the high Km (5.0 microM) enzymic activity of the membrane preparation nor the phosphodiesterase activities of the cytosolic fraction of the ADP-treated platelets was affected. This effect of ADP, which was independent of platelet aggregation, reached maximal level within 5 min of incubation. When platelet-rich plasma was incubated longer in the presence of nucleotide, the inhibition of phosphodiesterase activity began to decrease, and after 20 min of incubation approx. 90% of the original enzymic activity was regained. The incubation of platelet-rich plasma with 4.0 microM ADP also increased the cyclic AMP level to twice the basal level. The effect of ADP on the phosphodiesterase activity could be demonstrated only by incubating the intact platelets with the nucleotide. The treatment of isolated membrane from platelets, previously unexposed to ADP, with the nucleotide did not inhibit the enzymic activity. The inhibition of phosphodiesterase by the nucleotide in the absence of stirring, as expected, resulted in the inhibition of platelet aggregation when these cells were subsequently stirred with 1-epinephrine or an increased concentration of ADP.