1. The aspirin concentrations previously reported to inhibit platelet aggregation in vitro (40-500 mumol/l) are much greater than those required in vivo in man (5 mumol/l). 2. Human platelet-rich plasma was incubated with buffer or various aspirin concentrations at 37 degrees C for up to 4.5 h. Platelet aggregation and thromboxane generation were measured in response to collagen (0.4-6.3 micrograms/ml) and adenosine 5'-pyrophosphate (0.5-4 mumol/l). 3. The concentration of aspirin needed to inhibit platelet aggregation in response to a critical concentration of aggregating agent (lowest concentration to cause greater than 50% aggregation) was lower than that required for higher concentrations of aggregating agent. 4. With more prolonged incubation times with aspirin, lower concentrations of aspirin inhibited platelet aggregation. 5. Inhibition of platelet aggregation and thromboxane formation by 10 mumol/l aspirin was maximal by 90 min. There was progressive inhibition by 3 mumol/l aspirin during incubation for 270 min. By the end of this time there was also significant inhibition by 1 mumol/l aspirin. 6. The apparent discrepancy between inhibitory aspirin concentrations in vivo and those observed in vitro in previous studies appears to have been resolved by extending the incubation time of platelets with low aspirin concentrations, thus mimicking the conditions in vivo.