T he cDNAs for two separate human 17 beta-hydroxysteroid dehydrogenases (17 beta-HSD) have been isolated and sequenced. The well-studied human placental cytosolic 17 beta-HSD (also referred to as estradiol dehydrogenase) preferentially catalyzes the reduction of estrone to estradiol-17 beta and the reduction of the C-20-ketone of progesterone to 20 alpha-dihydroprogesterone. This isoform of the enzyme has been referred to as 17 beta-HSD type 1 and localized to chromosome 17. A second 17 beta-HSD isoform (referred to as type 2) is localized in the endoplasmic reticulum of human trophoblast and is characterized by the preferential oxidation of the C-17 beta-hydroxyl group of C18- and C19-steroids and the C-20 alpha-hydroxyl group of 20 alpha-dihydroprogesterone. In this study, we determined the chromosomal localization of human 17 beta-HSD type 2, the expression of this gene in human endometrium, and the tissue distribution of the mRNA. We found that the human 17 beta-HSD type 2 gene is localized on chromosome 16, 16q24. 17 beta-HSD type 2 mRNA (approximately 1.5 kb) was identified in human endometrial tissues by Northern analysis of total RNA (10 micrograms). The highest levels of 17 beta-HSD type 2 mRNA were found in endometrial tissues obtained during the mid- to late secretory phase of the ovarian cycle (i.e., during the time of high plasma levels of progesterone). 17 beta-HSD type 2 mRNA levels were much greater in glandular epithelium than in the stromal cells isolated from secretory phase endometrium. The levels of 17 beta-HSD type 2 mRNA in secretory phase endometrium were approximately one-tenth that in villous trophoblast tissue from human placenta. We did not detect 17 beta-HSD type 1 mRNA in endometrial tissue by Northern analysis of total (10 micrograms) RNA. These findings are consistent with the view that the progestin-regulated 17 beta-HSD of the glandular epithelium of the human endometrium is primarily, if not exclusively, the product of the 17 beta-HSD type 2 gene. 17 beta-HSD type 2 mRNA was present in human placenta, liver, and small intestine; much smaller amounts, barely detectable by Northern analysis of poly(A)+ RNA, were present in prostate, kidney, pancreas, and colon, but not in heart, brain, skeletal muscle, spleen, thymus, ovary, or testis.