Acid-soluble extract of human bladder mucosal surface was obtained by washing out the bladder of accidentally dead male human with 1% acetic acid in the presence of proteinase inhibitors. Acid urea polyacrylamide gel electrophoresis (AU-PAGE) indicated that the acid-soluble extract had more than 10 main protein bands, but the currently known antibiotic peptides, e. g., Lysozyme and defensins, were not found in this extract. The quantitation assay of lysozyme showed that lysozyme only amounted to 2.2 microgram per milligram protein. When tested for antibacterial activity by using ultrasensitive radial diffusion assay, the acid-soluble extract effectively killed E. coli ML-35P. The antibacterial activity of the acid-soluble extract was further analysed by gel overlay technique and the result showed that one main protein band, which was referred to as human bladder protein (HBP), was potently antibacterial against E. coli ML-35P. The HBP accounted for 4% of the total acid-soluble extract protein. These results suggest that the presence of antibacterial protein in the bladder mucosa may be the molecular base of the bladder antibacterial defence mechanisms.