Intact epithelial glands and stromal cells were isolated from normal proliferative and secretory endometrial specimens by digesting the tissue fragments with 0.1% collagenase solution. These components were identified by the structure of the glands and the distinct morphology of the cell cultures. The same morphological characteristics and estradiol dehydrogenase (E2DH) activity were maintained from generation to generation in the primary cell cultures derived from either glands or stroma. The generation time of cultured stromal cells was shorter than that of glandular epithelial cells. The growth rate of both cell cultures was accelerated by addition of estradiol (E2) and medroxyprogesterone acetate in culture medium. Metabolism of E2 was studied in the freshly isolated glands and stromal cells by incubating these components, with radioactive E2 or estrone (E1) at various concentrations and time intervals. Qualitative and quantitative variations in the metabolic pattern of E2 were distinguished in epithelial glands and stromal cells. Two major metabolities, E1 and E1 sulfate (E1S), were identified in an incubation of epithelial glands of secretory endometrium, while only E1 was found in stromal cells. The maximum rate of formation of E1 was much nigher than that of E1S in epithelial glands. At low E2 concentration, similar to that of plasma E2, the amount of E1S formed in glands was comparable to that of E1. At high E2 concentration, the formation of E1 increased linearly in proportion to the concentration of E2, whereas the formation of E1S reached a saturation level. More than 70% of E1 and 90% of E1S formed were released to the medium, suggesting that both metabolites facilitate the expulsion of the estrogen from these target cells. The conversion of E2 to E1 was significantly higher in both glands and stroma of the secretory endometrium compared to proliferative endometrium. The highest E2DH activity was found in the epithelial glands of the secretory phase, followed, in order, by the stromal cells in the secretory phase, the epithelial glands of the proliferative phase, and, finally, the stromal cells in the proliferative phase. Kinetic analysis of the E2DH activity indicated that both glands and stroma hr.ve the same type of enzyme.