The acid-soluble extract of the bladder mucosal surface was obtained by washing out the bladder with dilute acetic acid in the presence of protease inhibitors. The wash-out materials from rats, rabbits, pigs, and humans manifested strong bactericidal activity against E. coli in vitro. The ultrafiltrate of the human material, which contained two major peptides with apparent molecular masses of 6.7 kD and 8.5 kD, respectively, showed potent bactericidal activity against E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus sanguis. Three antibacterial polypeptides (PiBPs) were purified from the porcine material. The molecular masses of PiBP-5, PiBP-11 and PiBP-25 were 5773.3 Da, 11127.8 Da and 25073 Da, respectively. PiBP-5 was unusually rich in glycine, serine and threonine residues (20.0, 16.3 and 10.4 mol%, respectively), and N-terminal amino acid sequencing revealed that PiBP-5 was homologous (83.3% identity in an 18 residue overlay) to the" tail" of human cytokeratin-7. Although the amino acid compositions of PiBP-11 and PiBP-25 were established, both had blocked N-termini and primary sequence data were not obtained. These results provided evidence indicating that the presence of peptides in the bladder mucosa could enable it to kill adherent bacteria.