The hypothesis that neutrophils play an important role in the pathogenesis of gastric ulceration induced by nonsteroidal anti-inflammatory drugs (NSAIDs) was tested in rats. Rats made neutropenic by prior treatment with an antibody to rat neutrophils raised in goat were found to be significantly more resistant to the gastric-damaging actions of indomethacin or naproxen than were control rats or rats pretreated with normal goat serum. The reduction of damage in neutropenic rats was not due to effects of the antineutrophil serum on either gastric acid secretion or the ability of indomethacin or naproxen to inhibit prostaglandin synthesis. Gastric cyclooxygenase activity was inhibited by greater than 95% in both normal and neutropenic rats that received indomethacin or naproxen. Reduction of circulating neutrophil numbers by treating rats with methotrexate also resulted in a significant reduction in the susceptibility to gastric damage induced by indomethacin. Since activation of circulating neutrophils appeared to be important in the development of gastric erosions after administration of indomethacin, and in the significant changes in vascular endothelial integrity (Monastral Blue staining) observed within 15 min of indomethacin administration, we investigated the possibility that leukotrienes (LTs) and platelet-activating factor (PAF) might be involved in the pathogenesis of indomethacin-induced ulceration. Changes in gastric LTB4 synthesis were not observed after indomethacin administration. Pretreatment with either an LTD4 antagonist or a PAF antagonist was without significant effect on the extent of gastric damage induced by indomethacin. These results suggest an important role for neutrophils in the pathogenesis of NSAID-induced gastric ulceration. Neutrophils may be important in the vascular injury that occurs early after administration of these compounds.