The ototoxicity of gentamicin (GM) and Kanamicin (KM) was analyzed using an organ culture system. Inner ear of mouse 16 day embryos were cultured for 5 days with or without GM or KM. The damage of crista ampullaris and macula utriculi of cultured inner ear was evaluated according to our ototoxicity score based on the morphological change by a light microscopic observation of serial sections of the samples. We gave one grade damage for each of the following criteria; 1) damage of apical surface of the hair cells, 2) debris in the endolymph space, 3) disappearance of the hair cells, 4) degeneration of the supporting cells. Using this system we showed that the effect of GM and KM was dose dependent and that GM was more toxic than KM at the concentration of more than 100 μg/ml. This organ culture system can serve as a useful and convenient model system of ototoxic evaluation.