Adverse trends in the reproductive health of male fish, including testis abnormalities and intersex gonads, have been increasingly reported over recent years. These effects have been associated with the exposure of fish to natural, synthetic, and xenobiotic estrogens present in the aquatic environment. A novel in vivo test system using transgenic zebrafish has been developed to rapidly determine the effects of estrogenic chemicals on critical life stages and sensitive target organs in the fish. In the transgenic zebrafish, an estrogen binding sequence linked to a TATA box and luciferase reporter gene was stably introduced. Binding of a substance to endogenous estrogen receptors (ER) and the subsequent transactivation of the ER result in luciferase gene induction that is easily measured in tissue lysates. Exposure to estradiol (E2) during juvenile stages of the transgenic zebrafish revealed the period of gonad differentiation to be the most responsive early life stage. In adult male transgenic zebrafish, the testis was the most sensitive and responsive target tissue to estrogens. Partial sequences of zebrafish estrogen receptor subtypes α and β were cloned for the first time and were found to be differentially expressed in developing fish and tissues of adult male zebrafish. The transgenic zebrafish assay is a promising new tool to rapidly determine the estrogenic potency of chemicals in vivo.