Incubation of Escherichia coli (10(4)/ml) in cell-free rabbit lung lavage for 30 min at 37 degree C resulted in a 70% reduction in microbial counts on deoxycholate agar but no decrease on blood agar. This effect was not due to agglutination, and the order of exposure was important, i.e., activity was seen only if lavage incubation proceeded deoxycholate treatment. After high speed centrifugation of lung lavage (50,000 X g), activity remained in the supernatant and not in the surfactant pellet, Ultrafiltration of the supernatant (UM to filter) yielded an active ultrafiltrate and an inactive retent. Ultrafiltrate activity was unaffected by heating to 95 degrees C but could be removed by treatment with trypsin or bentonite. Sephadex G-15 fractionation of lyophilized ultrafiltrate yielded three active peptide peaks. Electron photomicrographs showed that incubation with the initial G-15 peak followed by deoxycholate resulted in the disappearance of intracellular material in about half the cells, a finding not seen with deoxycholate or peptide along, and EDTA reversed activity of the G-15 peptide and ultrafiltrate. Rabbit lung lavage contains a complex antimicrobial system that facilitates bile acid destruction of bacteria.