Pseudomonas aeruginosa strains isolated from patients with persistent lung infections and cystic fibrosis have been found to gradually develop aminoglycoside resistance over time. The aim of this study was to identify potential contributors to low-level aminoglycoside resistance, which may cause such graduated increases in resistance. The Harvard P. aeruginosa PA14 nonredundant library, consisting of approximately 5,800 mutants, was screened for twofold or greater increases in tobramycin resistance. Mutants carrying mutations in a total of 135 unique genes were identified and confirmed to have reduced susceptibility to tobramycin. Many of these genes were involved predominantly in energy metabolism; however, most of these mutants did not exhibit growth defects under the conditions tested, although some exhibited the small-colony phenotype and/or defects in growth under anaerobic conditions. Lipopolysaccharide mutants were also identified, and it was found that tobramycin had a reduced ability to permeabilize the outer membranes of these mutants. The results of this study emphasize the complexity of the interactions that tobramycin may have within the bacterial cell and introduce a large number of novel genes which may play a role in tobramycin resistance.