Three days post-parturition mosquitofish were exposed to different concentrations of 4-NP following a semi-static protocol. Exposure lasted up to the development of male anal fin in male individuals of the control group. Exposure to 50 μg/L 4-NP resulted in 100% females considering secondary sexual characters, while external sex-ratio did not statistically differed from unity in control group. In group exposed to 0.5 and 5.0 μg/L sex-ratio did not differ from unity but incompletely developed gonopodium was observed in several individuals. Individuals exposed to 50 μg/L 4-NP exhibited female or undeveloped gonads, while gonadal sex-ratio did not statistically differ from unity in control group. Percentage of undeveloped gonads increased with 4-NP concentration. Additional observations demonstrated hepatic histopathology in fish exposed to the highest concentration and growth reduction dependent on 4-NP concentration. In a complementary experiment, extensive metabolism of [3H]4-n-NP was characterized following in vivo exposure of juvenile mosquitofish suggesting that metabolism could modulate 4-NP toxicity. This study suggests susceptibility of early life stages of mosquitofish to endocrine modulators with regard to development of reproductive capabilities.