The aminoglycoside antibiotics are important agents in the treatment of bacterial infection. At pharmacological doses they also preferentially damage the sensory hair cells of the inner ear, leading to ototoxic hearing loss. However, it has been suggested that the mechanism of ototoxicity is different from that of bacterial toxicity. The bacterial neomycin phosphotransferase gene (Neo) confers resistance to this and related aminoglycoside antibiotics. To determine whether the Neo gene also confers resistance to vertebrate ototoxicity, the sensitivity of cochlear hair cells to neomycin was evaluated in mice with a targeted insertion of Neo. Organotypic cultures of the organ of Corti, isolated from neonatal wild-type mice and two strains of mice carrying the Neo gene, were cultured for 72 h in the absence (controls) or in the presence of 200 microM neomycin. Organs from wild-type mice showed no remaining outer hair cells and <25% of inner hair cells when incubated with neomycin. In contrast, organs from mice carrying the Neo gene showed no loss of hair cells after neomycin treatment.