Repeated transfers of strains of Escherichia coli and Staphylococcus aureus in medium containing subminimal inhibitory concentrations (sub-MICs) of gentamicin caused a moderate increase in the minimal inhibitory concentration of gentamicin. At the end of such transfers, E. coli K12 produced aminoglycoside phosphotransferase(3′)-I [APH(3′)], and E. coli R112, which carries the plasmid-coded enzyme APH(3 ′)-I, also produced the acetylating enzyme aminoglycoside acetyltransferase(2′) [AAC(2′)]. E. coli R148, which produces aminoglycoside phosphotransferase(3′)-II [APH(3′)-II), did not change its output of enzymes. Repeated transfers to media containing increasing concentrations of gentamicin resulted in the development of complete resistance to all aminoglycosides without the concurrent development of any demonstrable new enzyme activity. With repeated transfers in drug-free medium, a complete reversal to susceptibility to gentamicin, but not to other aminoglycosides, was obtained for strains that had previously been transferred in sub-MICs of gentamicin, whereas strains that had been transferred in increasing concentrations of gentamicin did not revert to their original sensitivity to aminoglycosides despite repeated transfers in drug-free medium.