Previous investigations have shown that ibuprofen inhibits the second wave of platelet aggregation and blocks the conversion of 14C-arachidonic acid to thromboxane. However, the influence of the drug on platelet function and cyclooxygenase is transitory, lasting only 24 hours. The present study has taken advantage of the short-lived influence of ibuprofen to study its interaction with the long-term effects of aspirin. As expected, both aspirin and ibuprofen suppressed platelet cyclooxygenase activity and function, but addition of aspirin to ibuprofen-treated platelets did not increase the degree of inhibition in vitro. Platelet function and prostaglandin synthesis recovered completely 26 hours following ingestion of ibuprofen, but remained compromised 26 hours after taking aspirin. When 650 mg of aspirin was administered after ibuprofen, platelet function and cyclooxygenase activity recovered as completely at 26 hours as did platelets which had been exposed to ibuprofen alone. Thus, prior exposure to ibuprofen in vivo completely protected cyclooxygenase from the irreversible effects of aspirin. Our findings indicate that ibuprofen-like indomethacin and other nonsteroidal antiinflammatory drugs react with the heme group of cyclooxygenase to prevent arachidonic acid conversion. Since ibuprofen completely blocks the effects of aspirin in platelets in vitro and in vivo, aspirin's primary influence on inhibition of cyclooxygenase must also be through action on the heme portion of the enzyme, rather than acetylation of the protein.