Acid-soluble extract of rabbit bladder mucosa was obtained by washing out the bladder of normal female New Zealand rabbit with 1% acetic acid in the presence of proteinase inhibitors. Acid urea polyacrylamide electrophoresis (AU-PAGE) analysis indicated that the extract had more than 10 main protein bands. The currently-known antibiotic peptides, e. g. lysozyme and defensin-like molecules were not found in this material. When tested for antibacterial activity by using ultrasensitive radial diffusion assay, the acid-soluble extract effectively killed E. coli ML-35P. The gel overlay assay showed that the anti-bacterial activity of the acid-soluble extract was relevant to two protein bands referred to as rabbit bladder protein 1 (Rab BP-1) and rabbit bladder protein 2 (Rab BP-2). The Rab BP-1 and Rab BP-2 accounted for 2.5% and 1.2% of the total acid-soluble extract proteins, respectively. These results suggest that the long recognized ability of the normal rabbit bladder wall to kill adherent E. coli may result from the presence of endogenous antibacterial proteins associated with its mucosa.