Platelet activation by the stable endoperoxide analogue U46619 is mediated largely by ADP released from platelet-dense granules. Polymorphonuclear leukocytes (PMNs) endowed with ecto-ADPase activity may operate as antiaggregatory cells in platelet aggregation induced by U46619. Unstimulated PMNs were effective in reducing aggregation when platelets were stimulated by threshold concentrations of U46619, whereas at higher concentrations of the stimulus, PMN activation is required. Evidence that the inhibition was mediated by PMN ecto-ADPase activity was obtained by high-performance liquid chromatography analysis, indicating that PMNs were able to efficiently metabolize platelet-active ADP into AMP. Moreover, PMN-derived supernatants were able to inhibit platelet aggregation, suggesting that under this circumstance the inhibition was exerted by an uncharacterized, releasable ADPase activity. This study supports the hypothesis that, besides nitric oxide and hydrogen peroxide, ADPase activity may represent another PMN-mediated pathway capable of regulating platelet activity in areas of reduced blood flow, such as those found in conditions of myocardial ischemia.