Polyclonal antibodies produced against human placental 17 beta-hydroxysteroid dehydrogenase (17HSD), purified to homogeneity, and the corresponding cDNA for the enzyme were used to study the expression of 17HSD in a number of human tissues using various immunological methods together with RNA hybridization techniques. In addition, two 17HSD genes and their putative regulatory elements were sequenced. Immunoblotting analysis showed that the placental-type enzyme is expressed in granulosa-luteal cells, breast cancer tissue and breast cancer cell lines. An immunologically identical antigen was also detected in normal and carcinomatous human endometrium. The same antiserum, following affinity purification, was used for immunohistochemical studies of the endometrium and breast tissue, whereupon staining of the cytoplasm of the epithelial cells alone was observed. Immunostaining was also present in cultured human granulosa cells and in about half of the endometrial and breast carcinoma specimens investigated. Progesterone induction of the 17HSD enzyme protein was demonstrated in the human endometrium during the secretory phase of the menstrual cycle and in one breast cancer cell line (T-47D) following progestin treatment. There are at least two mRNAs for placental 17HSD (1.3 kb, 2.3 kb). RNA hybridization analysis of various breast cancer cell lines showed that the 1.3 kb mRNA was most closely associated with enzyme protein expression and was also the only form responding to progesterone induction. We conclude that placental-type 17HSD is also expressed in some other human tissues, both steroid-synthesizing and steroid-responding, and that the mRNA and enzyme protein are induced by progesterone. The availability of the sequence of 17HSD genes and surrounding regions allows us to study the sequences responsible for the expression and regulation of 17HSD.