Functional in vitro and in vivo reporter gene assays have recently been developed for the rapid determination of exposure to (xeno)estrogens. The in vitro estrogen receptor (ER)-mediated chemically activated luciferase gene expression (ER-CALUX) assay uses T47D human breast cancer cells stably transfected with an ER-mediated luciferase gene construct. In the in vivo assay, transgenic zebrafish are used in which the same luciferase construct has been stably introduced. In both assays, luciferase reporter gene activity can be easily quantified following short-term exposure to chemicals activating endogenous estrogen receptors. The objective of this study was to compare responses by known (xeno)estrogenic compounds in both assays. Exposure to the (xeno)estrogens estradiol (E2), estrone, ethynylestradiol (EE2), o,p′-DDT, nonylphenol (NP), and di(2-ethylhexyl)phthalate (DEHP) revealed that EE2 was the most potent (xeno)estrogen tested and was 100 times more potent than E2 in the transgenic zebrafish assay, whereas in the in vitro ER-CALUX assay, EE2 and E2 were equipotent. Although the xenoestrogens o,p′-DDT and NP were full estrogen agonists in the in vitro ER-CALUX assay, only o,p′-DDT demonstrated weak dose-related estrogenic activity in vivo. To determine if differences in reporter gene activity may be explained by differential affinity of (xeno)estrogens to human and zebrafish ERs, full-length sequences of the zebrafish ER subtypes α, β, and γ were cloned, and transactivation by (xeno)estrogens was compared to human ERα and ERβ. Using transiently transfected recombinant ER and reporter gene constructs, EE2 also showed relatively potent activation of zebrafish ERα and ERβ compared to human ERα and ERβ. Zebrafish ERβ and ERγ showed higher transactivation by (xeno)-estrogens relative to E2 than human ERβ.