Pseudomonas aeruginosa is inherently resistant to most conventional antibiotics. The mechanism of resistance of this bacterium is mainly associated with the low permeability of its outer membrane to these agents. We sought to assess the bactericidal efficacy of liposome-entrapped aminoglycosides against resistant clinical strains of P. aeruginosa and to define the mechanism of liposome-bacterium interactions. Aminoglycosides were incorporated into liposomes, and the bactericidal efficacies of both free and liposomal drugs were evaluated. To define the mechanism of liposome-bacterium interactions, transmission electron microscopy (TEM), flow cytometry, lipid mixing assay, and immunocytochemistry were employed. Encapsulation of aminoglycosides into liposomes significantly increased their antibacterial activity against the resistant strains used in this study (MICs of >32 versus /ml). TEM observations showed that liposomes interact intimately with the outer membrane of P. aeruginosa, leading to the membrane deformation. The flow cytometry and lipid mixing assays confirmed liposome-bacterial membrane fusion, which increased as a function of incubation time. The maximum fusion rate was 54.3% 1.5% for an antibiotic-sensitive strain of P. aeruginosa and 57.8% 1.9% for a drug-resistant strain. The fusion between liposomes and P. aeruginosa significantly enhanced the antibiotics' penetration into the bacterial cells (3.2 2.3 versus 24.2 6.2 gold particles/bacterium, P < 0.001). Our data suggest that liposome-entrapped antibiotics could successfully resolve infections caused by antibiotic-resistant P. aeruginosa through an enhanced mechanism of drug entry into the bacterial cells.