Three strains of Pseudomonas aeruginosa resistant to gentamicin obtained as representative gentamicin-resistant clinical isolates from the University of Alberta Hospital (UAH) in Edmonton, Canada were characterized to determine their mechanism of resistance. All strains showed wide aminoglycoside resistance (tobramycin, sisomicin, amikacin, streptomycin, kanamycin, SCH 20569) but contained no evidence of gentamicin-acetylating, adenylating or phosphorylating activity. Gentamicin inhibited amino-acid incorporation in cell-free systems equally well with either ribosomes or soluble cell fractions obtained from either resistant or sensitive strains. Plasmid DNA was detected in two strains but resistance could not be transferred by conjugation to either P. aeruginosa or Escherichia coli recipients. The resistant strains showed a marked reduction in energy-dependent accumulation of gentamicin compared to a sensitive strain. These strains which are common at UAH are most likely resistant due to a failure of gentamicin to be transported across the cytoplasmic membrane to ribosomal sites until relatively high external gentamicin concentrations.