The acid-soluble extract of rat uterus cavity wall was obtained by washing out the normal Sprague-Dawley rat uterus cavity with 1% acetic acid in the presence of protease inhibitors. When tested for antibacterial activity by sensitive agarose diffusion assay, the extract effectively killed E. coli ML-35P. The bactericidal activity of the acid-soluble extract was further analyzed by gel overlay technique. The result showed that three protein bands, which were designated rat uterus protein 1 (rat UP-1), rat uterus protein 2 (rat UP-2), and rat uterus protein 3 (rat UP-3), were potently antibacterial against E. coli ML-35P. The rat UP-1, rat UP-2, and rat UP-3 accounted for 4.5%, 5.7%, and 6.6% of the total proteins of the rat uterus acid-soluble extract respectively. The lysoplate assay for the determination of lysozyme activity showed that extract had some lysozyme activity, but the activity was very low. AU-PAGE analysis of the extract did not show the presence of lysozyme on the gel. This study suggested that the uterus wall might secrete some currently-unknown antibiotic polypeptides which play an important role in the uterus defense against bacterial infections.