Estrogen metabolism was studied in a newly established cell line (RL95-2) derived from a human endometrial carcinoma. Estradiol and estrone were metabolized to water-soluble derivatives by cells under in vitro culture conditions. Between 80-90% of the added steroids were metabolized, with nearly quantitative recovery of the products from the incubation medium. Arylsulfatase treatment converted the metabolites to ether-soluble forms, whereas beta-glucuronidase had no effect on the aqueous solubility of these compounds. Butanol extracts of the water-soluble estradiol metabolites cochromatographed on high performance liquid chromatography with 17 beta-estradiol-3-sulfate (93.6%) or estrone-3-sulfate (3.5%). No more than 6% of the estradiol added to the incubation medium was recovered in the form of estrone, either as estrone or estrone sulfate. After arylsulfatase treatment of the estradiol conjugates, 92% of the ether-soluble radioactivity cochromatographed with estradiol, and 3.8% cochromatographed with estrone. Estrogen-sulfurylating activity was localized in the cytosol of subcellular fractions of RL95-2 cells. The sulfoconjugation of estrogens by RL95-2 cells may prove useful as a model for the investigation of estrogen metabolism in endometrial carcinoma cells.