The lipopolysaccharide (LPS) of Pseudomonas aeruginosa PAO1 possesses two distinct types of O polysaccharide, A and B band LPSs, but the majority of clinical isolates from cystic fibrosis patients who are infected with the organism possess only the A band as the major LPS antigen. The initial step in a series of events during the uptake of aminoglycoside antibiotics such as gentamicin is the ionic binding of the molecule to the cell surface. In an attempt to elucidate the role of A and B band LPSs of P. aeruginosa in this passive ionic binding of gentamicin to the outer membrane and its possible lethal effects, strains PAO1 (A+B+) and LPS isogenic derivatives (A+B-,A-B+,A-B-) were treated with the antibiotic. Ionic binding of gentamicin appeared to be subtly different in PAO1 and its LPS derivatives; a lethal dose of drug was bound to all strains, although the degree of binding varied with each strain. The outer membrane affinity for gentamicin was higher in strains possessing the B band than in strains with A band LPS, and these B band strains were more prone to antibiotic-induced killing. Strains with both A and B band LPSs bound the most gentamicin of all strains, and this binding caused an almost 50% loss in viability. Ionic binding of aminoglycoside antibiotucs to the outer membrane of cell surfaces must not only weaken th cell surface (R. E. W. Hancock, Annu. Rev. Microbiol. 38:237-264, 1984; N. L. Martin and T. J. Beveridge, Antimicrob. Agents Chemother. 29:1079-1087, 1986; S. G. Walker and T. J. Beveridge, Can. J. Microbiol. 34:12-18, 1988) but it must also be more important in cell death than was originally thought.