Honeybee (Apis mellifera) are frequently exposed to and likely to be infected by plant-associated bacteria. We mimicked this process by injecting bees with live bacteria and isolated five induced antibacterial substances by comparative liquid chromatographic mapping of the hemolymph. Three of these antibiotics belong to a unique family of small (18 amino acids) peptides: the apidaecins [Casteels et al. (1989) EMBO J. 8, 2387-2391]. We have now characterized a fourth bee immune response peptide. The complete sequence was established by Edman degradation of the peptide and fragments thereof. It is 34 amino acids long and contains 10 proline residues. The amino-terminal half is related to the apidaecins; similar proline motifs are also present in the amino-terminal quarter of the much longer fly diptericins. The newly identified peptide's broad spectrum, lower specific activities against Gram-negative plant pathogens and its inability to inhibit bacterial growth at medium ionic strength are different from the apidaecins. Moreover, the highest observed specific activity was against an apidaecin-resistant Xanthomonas strain. In contrast to the immediate action of apidaecins, bactericidal activity is delayed. We propose the name 'abaecin' for this new antibacterial response peptide.