Endometrial glands were separated from stromal cells by collagenase digestion of human endometrium, and the distribution of progesterone (P) receptor and the activities of P-regulated enzymes, 17 beta-estradiol dehydrogenase (E2DH) and 20 alpha-dihydroprogesterone dehydrogenase (20 alpha DH), were determined in these preparations. Concentrations of cytosolic P receptor were estimated by Scatchard plot analysis of specific [3H]P binding in intact proliferative endometrium and in glands and stromal cells isolated from this tissue. Epithelial cells were more than 10-fold enriched in high affinity, P-specific binding sites compared to stromal cells. The binding constants (Kd) for [3H]P binding were essentially similar in undissociated endometrium, glandular epithelium, and stroma, ranging from 1--5 nM. The activities of E2DH and 20 alpha DH were also 3-fold higher in the glandular epithelium than in whole tissue or stroma during both proliferative and secretory stages of the menstrual cycle. In addition, the induction of these enzyme activities by progestin in vitro in cultured explants of proliferative endometrium was restricted to the glandular epithelium. Thus, the effects of P on E2DH and 20 alpha DH are expressed in the same cells that contain receptors for P.