Background: Aspirin circulates transiently in blood, but the identity of the enzyme(s) that hydrolyzes its acetyl residue remains unknown. Results: Purification, mass spectrometry, and overexpression identified erythrocyte type I PAF acetylhydrolase as aspirin hydrolase. Conclusion: Aspirin is primarily hydrolyzed within erythrocytes by PAF acetylhydrolase. Significance: PAF acetylhydrolase and aspirin hydrolysis varies among individuals to modulate the effectiveness of aspirin.