An acid extract of human neutrophil granules was fractionated on a Sephadex G-100 column and tested for microbicidal activity against Acinetobacter calcoaceticus HO-1 as described previously (M.C. Modrzakowski and C. M. Paranavitana, Infect. Immun. 32:668-674, 1981). The low-molecular-weight protein fraction, peak D, accounted for about 30% of the protein and nearly all of the activity of the crude extract against strain HO-1. Peak D protein proved to be a mixture of the three human defensin peptides HNP1, HNP2, and HNP3. Purified defensins reproduced the microbicidal activity of peak D. The data suggest that defensins could play a major role in the killing of A. calcoaceticus by human neutrophils.