The apramycin-resistant mutants of E. coli were isolated spontaneously. Mutations, aprA and aprB, conferring resistance to apramycin were located at 72 min on the E. coli genetic map where most of ribosomal genes were mapped. One of the mutants, carrying the aprB mutation, has an altered translational fidelity expressed as severe restriction of amber suppressor activity in vivo. The chromosome location and altered translational fidelity indicate that the apramycin-resistant phenotype could be a consequence of an alteration of the ribosomal structure.