The enzyme 3beta-hydroxysteroid dehydrogenase/isomerase (3betaHSD) is required for the biosynthesis of all active steroid hormones. It exists as multiple isoforms in humans and rodents, each a product of a distinct gene. Two isoforms, 3betaHSD I and II, are expressed in a tissue-specific manner in humans. 3betaHSD I is the only isoform expressed in the placenta, where it is required for the biosynthesis of progesterone and thus essential for the maintenance of pregnancy. We recently identified two transcription factors, activating protein-2gamma (AP-2gamma) and the homeodomain protein, distaless-3 (Dlx-3), that are expressed in both human and mouse trophoblast cells that were shown to be required for trophoblast-specific expression of the orthologous murine 3betaHSD, 3betaHSD VI. Although we identified specific binding sites for AP-2gamma and Dlx-3 in the distal promoter of the human 3betaHSD I gene, HSD3B1, it was found that these transcription factors were not involved in determining placental-specific expression of human 3betaHSD I. Instead, a 53-bp placental-specific enhancer element located between -2570 and -2518 of the HSD3B1 promoter was identified. Within this 53-bp element, two potential placental transcription factor binding sites were found. EMSAs with a 20-bp oligonucleotide containing these two potential placental-specific binding sites identified one of the binding sites specific for the transcription enhancer factor (TEF)-5, which is highly expressed in human placenta and in placental choriocarcinoma-derived JEG-3 cells and the other overlapping binding site, specific for a GATA-like protein. Site-specific mutations in either the TEF-5 binding site or in the GATA binding site, each resulted in complete loss of enhancer activity. The data indicate that TEF-5 and the GATA-like protein act in a coordinate manner to determine the placental-specific expression of the human 3betaHSD I enzyme and therefore are critical for placental progesterone production required for the maintenance of pregnancy.