The sensitivity or resistance of gram-negative bacteria to antibacterial systems appears to be related to the length of the saccharide chain of the bacterial envelope lipopolysaccharides (LPS). To explore this relationship further, we made use of two bactericidal, membrane-active cationic proteins, recently purified to near homogeneity, one from human and one from rabbit polymorphonuclear leukocytes (PMN). We have studied the effects of these two closely similar proteins on strains of Salmonella typhimurium and Escherichia coli, each separate strain differing in the saccharide chain length of its outer membrane LPS. Binding of these proteins to the bacterial outer membrane is required for killing, and is accompanied by an almost immediate increase in outer membrane permeability to normally impermeant actinomycin D. Sensitivity to the bactericidal and permeability-increasing activities of the human and rabbit proteins increases with decreasing LPS-saccharide chain length (chemotype: [S < Ra < Rb(3) < Rc < Rd(1)]). S. typhimurium G-30 and E. coli J5, mutant strains lacking UDP-galactose-4-epimerase, synthesize incomplete LPS (chemotype Rc) when grown without galactose, and are then as sensitive to both PMN proteins as the S. typhimurium strains 395 R10 (Rd(1)) and R5 (Rb(3)). However, when these mutants are grown with galactose, they synthesize complete LPS (chemotype S) and exhibit nearly the same relative insensitivity as the smooth strains S. typhimurium 395 MS and E. coli 0111:B4. The differences among strains in sensitivity to the effects of the proteins on bacterial viability and permeability correspond to differences in bacterial binding of these PMN proteins. Thus, at protein concentrations that produce maximal antibacterial activity toward the rough bacteria, but little or no activity toward the smooth strains, rough bacteria bind from 3- to 10-fold more protein (S. typhimurium 395 R10; S. typhimurium G-30, and E. coli J5 [grown without galactose]) than do the smooth bacteria (S. typhimurium 395 MS; E. coli 0111:B4; S. typhimurium G-30 and E. coli J5 [grown with galactose]). These findings suggest that bacterial sensitivity or resistance to these purified bactericidal PMN proteins is determined by the binding properties of the outer membrane, which in turn depends upon the LPS-saccharide chain length.