Burkholderia cenocepacia (formally a genotype of Burkholderia cepacia complex called genomovar III) has emerged as a serious opportunistic pathogen in individuals with cystic fibrosis. We developed a liposomal antibiotic formulation composed of 1,2-distearoyl-sn-glycero-3-phosphocholine and cholesterol (molar ratio 2:1) to overcome B. cenocepacia's resistance to commonly used aminoglycosidic antibiotics.The dehydration-rehydration vesicles technique was used to entrap antibiotics in liposomes. The size of liposome formulations was measured and encapsulation efficiencies were determined by microbiological assays. MICs of free and liposomal antibiotics against the clinical isolates of B. cenocepacia were determined by the standard broth dilution method and in vitro time--kill studies were performed using free and liposomal antibiotics at one, two or four times the MICs. We studied the mechanism of action of these formulations by using transmission electron microscopy (TEM), fluorescence-activated cell sorter (FACS) analysis, lipid-mixing assay and immunocytochemistry.The encapsulation efficiencies of amikacin, gentamicin and tobramycin into liposomes were 52.08 +/- 5.4%, 27.72 +/- 1.14% and 28.08 +/- 1.54%, respectively. The liposome formulations were more stable in PBS at 4 degrees C than in PBS, bronchoalveolar lavage fluid or plasma at 37 degrees C. The TEM studies along with lipid-mixing assays and FACS analysis indicated the lipid contact of the liposomal bilayers and bacterial cell membranes. Most importantly, our liposomal formulations reduced MICs for highly antibiotic-resistant strains and enhanced the antibiotics' penetration into the bacterial cells. For instance, bacterial eradication by liposomal tobramycin was 4-fold higher than free tobramycin.A liposomal drug delivery system might enhance the efficacy of commonly used aminoglycosides.