17β-Hydroxysteroid dehydrogenase (HSD) was studied in endometrium removed surgically from intact female monkeys in the luteal phase of the menstrual cycle. Also castrated monkeys (N = 20) implanted with estradiol (E2) and progesterone (P) in silastic capsules were used to investigate the hormonal dependence of this enzyme in endometrium. The activity was characterized by an optimal pH between 9 and 10, a Km for E2 of 14.2 × 10-6M, a Vmax of 0.9 × 10-6M × min-1 in Tris-HCl buffer 50 mM pH 9. Using these conditions, we found linearity with time and protein concentration. Subcellular fractionation of endometrial homogenate showed a maximal activity in the 105,000 × g ultracentrifugation microsomal pellet and a very low activity in cytosol. HSD activity increased slightly, but distinctly, with E2 capsules alone, and a marked HSD stimulation (10 fold) occurred after P administration in combination with E2. There are strong similarities between this enzyme and the one described in human endometrium. © 1979.