Endometrial cells from the hamster's uterus were grown as a monolayer during primary cell cultures and the progesterone receptor synthesis in the cultured cells was determined. The cells were found to be estrogen responsive. Production of the progesterone receptor in the cultured cells was enhanced by the presence of 17 beta-estradiol at physiological concentrations. A significant increase in specific binding of (3H) R5020 was observed after 24h of culture; by 48 h, a 2- to 3-fold augmentation in binding was noted. The increase in the progesterone receptor depended on the concentration of estradiol, with maximal response at about 1 nM. The progesterone receptor response was specific for estrogen. Testosterone or cortisol induced small or no response on the production of progesterone receptors. The progesterone receptor synthesis was inhibited by cycloheximide (10 micrograms/ml) or actinomycin D (10 micrograms/ml). These results demonstrate that endometrial cell cultures can serve as a good in vitro system for the study of hormonal effects on progesterone receptor synthesis or other biochemical responses. We conclude that the progesterone receptor formation in the cultured endometrial cells is mediated via RNA and protein synthesis.