An attempt was made in vitro and in vivo to clarify the significance of aminoglycoside-resistant subpopulations that can be isolated from gentamicin-susceptible Pseudomonas aeruginosa. Population analyses revealed bacterial subpopulations resistant to greater than or equal to 8 micrograms/ml gentamicin in 23 of 24 gentamicin-susceptible strains tested. These subpopulations could easily be selected in a single exposure of high inocula of P. aeruginosa to two times the MIC of gentamicin for 24 to 48 hr. The increased gentamicin resistance was associated with slower growth and formation of smaller colonies. In serial subcultivations, most of the selected variants were unstable regarding gentamicin resistance and colony morphology. Stable SCV representing the extreme form of gentamicin-resistant subpopulations were further studied. In comparison to the corresponding PS, they were fourfold to sixfold less susceptible to five aminoglycosides tested; their susceptibility to eight antipseudomonal beta-lactam drugs, however, was usually retained. SCV proved to be less pathogenic than PS for normal and moderately leukopenic mice. Agranulocytic mice, in contrast, were invariably killed after i.p. injection of less than 20 organisms of PS or SCV, although death occurred 24 to 48 hr later in mice infected with SCV. After injection into the thigh muscle of agranulocytic mice, SCV were not affected by therapeutic plasma levels of gentamicin, whereas ticarcillin was highly effective. Aminoglycoside-resistant subpopulations of P. aeruginosa could contribute to the high number of treatment failures of Pseudomonas injections in agranulocytic patients and may account, in part, for the superiority of combined antipsuedomonal chemotherapy in this clinical situation.