Aspirin, one of the most widely used drugs in the world, consistently produces gastric mucosal injury, but the pathogenic mechanisms are incompletely understood. The present study was designed to determine the role of neutrophils in aspirin-induced acute gastric mucosal injury. Gastric mucosal lesions induced by acidified aspirin (300 mg/kg) were completely prevented in rats that had been rendered profoundly neutropenic by anti-neutrophil serum. Aspirin-induced acute gastric mucosal lesions were also significantly, albeit incompletely, reduced in rats that had been rendered moderately neutropenic by methotrexate. Moreover, in the methotrexate-induced neutropenia model, the neutropenia-associated mucosal protection against aspirin-induced injury could be reversed by leucovorin rescue. Aspirin caused a marked and statistically significant reduction in gastric mucosal 6-ketoprostaglandin F1 alpha synthesis, but no significant changes in gastric mucosal leukotriene synthesis. Thus no gastric mucosal lesions were observed in profoundly neutropenic rats that were treated with aspirin, despite the marked inhibition of prostaglandin synthesis. These findings demonstrate that aspirin-induced acute gastric mucosal injury is a neutrophil-dependent process.