The purpose of this study is to investigate the ototoxic effect of streptomycin on cochlear hair cells, spiral ganglion cells, and nerve fibers in cochlear organ cultures. The cochlear basilar membrane of three- or four-day-old F344 rats was cultured and treated with various doses of streptomycin for 24 h. Cochlear hair cells and the spiral ganglion neurons were stained with immunofluorescence and were observed under confocal microscope. The increase in hair cell loss was concomitant with the increase in streptomycin sulfate concentrations. Streptomycin impaired both the inner and outer hair cells at a similar degree. The damage to hair cells was more severe at basal turn than apical turn. In contrast, the spiral ganglion neurons and auditory nerve fibers were intact. Streptomycin primarily caused hair cell loss, but not significant impairment in the neural structure in vitro. Streptomycin-induced cochlear hair cell lesion was initiated at the basal turn and extended towards the apical turn. Streptomycin ototoxicity was dose-dependent under in vitro conditions.