Indomethacin produces mucosal lesions in both the gastric antrum and small intestine in rats refed for 1 h after a 24-h fast. This study was designed to determine the role of bacteria in the formation of the antral lesions. A mixture of antibiotics (bacitracin, neomycin, and polymyxin B) prevented the antral lesions as well as intestinal lesions. The antibiotics also decreased the gastric corpus lesions induced by indomethacin in the fasted rat. Under a germ-free condition, indomethacin did not produce severe lesions in the small intestine of the refed rat but provoked many lesions in the antrum of the refed rat and in the corpus of the fasted rat. Corpus lesions induced by indomethacin in the fasted rat were decreased markedly by neomycin and slightly by polymyxin B, but not by bacitracin. Corpus lesions produced by an absolute ethanol, however, were prevented by each of the antibiotics. The inhibitory effect of neomycin on the corpus lesions was not blocked by pretreatment with indomethacin. In pylorus-ligated rats, neomycin did not decrease gastric acid secretion. The concentration of nonprotein sulfhydryls in the gastric mucosa was not altered by the treatment with neomycin. The antibiotic solutions were hypotonic. It is concluded that (a) bacteria are not important in the formation of antral and corpus lesions induced by indomethacin, and (b) antibiotics prevent gastric ulceration not by its antibacterial action, but by a "cytoprotective" action. The mechanism is unknown, but it may be different from that of antisecretory drugs, prostaglandins, mild irritants, hypertonic solutions, and sulfhydryl compounds.