Abstract We examined the effect of tributyltin (TBT) on the sex differentiation process in genetically female Japanese flounder (Paralichthys olivaceus). The fish were fed an artificial diet containing tributyltin oxide (TBTO) at concentrations of 0.1 and 1.0 μg/g diet from 35 to 100 d after hatching, which includes the sex differentiation period. The ratio of sex-reversed males significantly increased to 25.7% of the flounder fed the 0.1 μg/g diet and to 31.1% of those fed the 1.0 μg/g diet compared with the control (2.2%). From morphological and histological examination of the fish in the TBT-treated groups, normal females had typical ovaries and sex-reversed males had typical testes. These results clearly demonstrated the masculinization of flounder exposed to TBTO. This is the first report of TBT inducing sex reversal in vertebrates.