A sensitive ex vivo bioassay for detecting environmental estrogens and estrogen mimics was developed using Xenopus liver slice culture. Vitellogenin synthesis by these cultures was used as biomarker for estrogenic activity of environmental water samples. Sensitization of the assay for estrogens and mimics was accomplished by employing tissue from animals after in vivo preexposure to estrogen. Effects of various tissue culture factors were investigated in order to obtain optimum conditions for the bioassay. It was found that endogenous vitellogenin and/or estrogen could be “washed out” of cultures and that not only uncontaminated males but also estrogen-pretreated males and females can successfully be used as bioindicators. Estrogenicity was detected, using the ex vivo Xenopus liver slice culture assay, in sewage effluent, lake water, and dam water. This article presents an optimized protocol for effective estrogen detection in environmental water samples.