Despite undoubtful clinical evidence of anti-platelet effects of aspirin, previous studies demonstrate little inhibition by aspirin treatment of single platelet activation as measured by flow cytometry. This discrepancy was further evaluated using flow cytometric measurements of circulating platelet-leukocyte aggregates (PLAs), which may be a more sensitive marker of platelet activation in vivo than measurements of activation markers on single platelets. Blood samples were obtained from 15 healthy subjects before and after aspirin treatment (75 and 500 mg daily for 1 week). Platelet (P-selectin expression) and leukocyte (CD11b expression) activation and platelet-leukocyte aggregation were monitored by whole blood flow cytometry. Approximately 1% platelets in unstimulated samples were P-selectin-positive, i.e., circulating activated platelets, and about 3% of leukocytes were circulating as PLAs; neither of these parameters were reduced by aspirin treatment. Circulating platelet micro-aggregates were not influenced by aspirin either. In vitro stimulation with ADP, thrombin, or PAF increased platelet P-selectin expression and thus PLA formation, but these responses were not affected by aspirin. Leukocyte CD11b expression, a marker of leukocyte secretion, was not significantly influenced by aspirin either in unstimulated samples or upon in vitro stimulation. Thus, the present data support the concept that thromboxane generation is of little importance for the activation of single platelets; the platelet inhibiting effect of aspirin is seen mainly in the presence of close cell-cell contact, which enhances the importance of thromboxane. Multiple mechanisms may contribute to the remarkable clinical anti-thrombotic effect of aspirin.