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BACKGROUND Hemoglobin is a precursor of antibacterial peptides. Our aim was to identify an antibacterial peptide in human endometrium. We tested the antimicrobial activities of hemoglobin and a derived peptide in vitro and in vivo in rats. METHODS Samples (n = 3) were scraped from the surface of endometrium. Acid-soluble proteins underwent electrophoresis followed by gel overlay assay and reversed-phase high-performance liquid chromatography. Antibacterial activities were determined by agar radial diffusion assay. Purified peptides were further characterized by electrophoresis, mass spectrometry, N-terminal amino acid (AA) sequencing and protein structure analysis. A rat model was used to test the inhibitory activity of human hemoglobin on vaginal infection with Escherichia coli, using one experimental group (intravaginal hemoglobin, n = 9) and three control groups (n = 14). Vaginal histology was studied. RESULTS The purified peptide exhibited potent antibacterial activities against E. coli ML-35P. The N-terminal AA sequence was F L S F P T T K T Y, identical to AA 32–41 of the human hemoglobin α chain, and it had the same mass (m/z = 6776.8) as the α chain 32–93 AA fragment, with at least three α-helices. Histology indicated that the hemoglobin group changed significantly compared with the matrix control group (no treatment after infection): the surface layer of stratified squamous epithelium was smoother, inflammatory cell infiltration was relieved in the lamina propria and congestion pattern was decreased. CONCLUSIONS These results suggest that erythrocytes from endometrium are another source of the antimicrobial molecules. Hemoglobin and its derived peptides may play a role in the host defense against pathogens in human vagina. [ABSTRACT FROM AUTHOR]