Aspirin inhibits platelet function by permanently acetylating the cyclooxygenase that forms prostaglandins. We determined the sensitivity of platelets to aspirin in normal subjects by measuring [3H-acetyl]aspirin-susceptible cyclooxygenase in washed platelets obtained at various times after aspirin ingestion. A single 325-mg aspirin dose inactivated 89% of platelet cyclooxygenase. The inhibition persisted for 2 days suggesting that oral aspirin also inactivated megakaryocyte cyclooxygenase. Thereafter, active enzyme returned with a time-course reflecting platelet turnover (life-span 8.2+/-2 days). Single doses of 20-650 mg aspirin resulted in 34- greater than 95% inhibition after 24 h. Daily doses of 20-325 mg aspirin for brief periods produced 61- greater than 95% inactivation when measured 24 h after cessation of the drug. Platelet cyclooxygenase is more sensitive to inactivation by aspirin than enzyme in sheep seminal vesicles.