The phosphodiesterase (PDE) inhibitors AY-31,390, milrinone and pelrinone (AY-28,768) were analyzed in human platelet aggregatory systems and in a rabbit arteriovenous shunt model to delineate their activity. AY-31,390 showed a remarkably potent capacity to inhibit human antithrombotic platelet aggregation. AY-31,390 inhibited arachidonic acid, U46619, collagen, epinephrine (second phase) and adenosine diphosphate (second phase) induced platelet aggregation (PA) with IC50 values of 0.18, 0.21, 0.54, 0.43 and 0.20 microM, respectively. Milrinone, although less potent than AY-31,390, inhibited PA with IC50 values of 2.1, 2.0, 5.4, 3.7 and 4.1 microM and pelrinone's IC50 values were 2.8, 6.6, 13.3, 18.6 and 11.8 microM, respectively. Platelets which were incubated with AY-31,390, milrinone or pelrinone, washed with Hanks' balanced salt solution and then resuspended in platelet poor plasma, lost their inhibitory activity in collagen and arachidonic acid PA systems. These results suggested that AY-31,390, milrinone and pelrinone did not bind tightly to cAMP PDE. If human platelet-rich plasma was pretreated with adenosine deaminase, an enzyme that degrades adenosine, the inhibitory effect of milrinone and to a lesser extent pelrinone was reversed. AY-31,390 did not produce a loss of activity with adenosine deaminase in the arachidonic acid system and only a small loss in the collagen system. Adenosine did not appear to be a meaningful factor in AY-31,390's inhibitory activity. Pelrinone, milrinone to a greater extent, and AY-31,390 to the greatest extent were effective inhibitors of white thrombus formation in the in vivo rabbit arteriovenous shunt model. These PDE III inhibitors were potent deterrants of platelet aggregation and white thrombus formation; these agents would be expected to be efficacious therapeutic antithrombotics.