Objective To investigate the relationship between [Ca2 +]i and gentamicin ototoxicity. Methods Isolated outer hair cells were loaded with [Ca2 +]i - sensitive fluorescence dye Fluo- 3, and confocal laser microscopy was used to determine [Ca2 +]i when K +- Hanks、gentamicin or its metabolite were added. Results Gentamicin decreased [Ca2 +]i in short time, and inhibited inward calcium current induced by K + . On the contrary, metabolized gentamicin increased [Ca2 + ] i for a long period. It did not effect calcium current induced by K + . Conclusion Metabolized gentamicin increased [Ca2 + ]i, which may result in its ototoxicity.