Antibacterial factors were purified from human adenoid glands by tissue extraction and consecutive steps of reversed-phase chromatography and assayed for bactericidal activity against the airway pathogen Moraxella catarrhalis and also Escherichia coli and Bacillus megaterium. One of the most active components isolated from adenoids was identified by N-terminal sequence analysis and mass spectrometry as high mobility group box chromosomal protein 1 (HMGB1). This novel finding was further substantiated by Western blot analysis, demonstrating a protein of expected size reactive with HMGB1 antiserum. Local synthesis was confirmed by reverse-transcriptase PCR and in situ hybridization. Adenoid-derived HMGB1 and recombinant HMGB1 revealed comparable antibacterial activity at high rate. More than 95% of bacteria were eradicated within 5 min by HMGB1 in the cultures. Secretion from the adenoid gland surface was also demonstrated to contain antibacterial activity, mainly mediated by alpha-defensins, but not by HMGB1. We conclude that HMGB1, produced and stored intracellularly in the adenoid gland, contributes to the local antibacterial barrier defense system in the upper respiratory tract.