The single-copy gene encoding the a subunit of glycoprotein hormones is expressed in the pituitaries of all mammals and in the placentas of only primates and horses. We have systematically analyzed the promoterregulatory elements of the human and bovine a-subunit genes to elucidate the molecular mechanisms underlying their divergent patterns of tissue-specific expression. This analysis entailed the use of transient expression assays in a chorionic gonadotropin-secreting human choriocarcinoma cell line, protein-DNA binding assays, and expression of chimeric forms of human or bovine a subunit genes in transgenic mice. From the results, we conclude that placental expression of the human a-subunit gene requires a functional cyclic AMP response element (CRE) that is present as a tandem repeat in the promoter-regulatory region. In contrast, the promoter-regulatory region of the bovine a-subunit gene, as well as of the rat and mouse genes, was found to contain a single CRE homolog that differed from its human counterpart by a single nucleotide. This difference substantially reduced the binding affinity of the bovine CRE homolog for the nuclear protein that bound to the human a CRE and thereby rendered the bovine a-subunit promoter inactive in human choriocarcinoma cels. However, conversion of the bovine a CRE homolog to an authentic a CRE restored activity to the bovine a-subunit promoter in choriocarcinoma cells. Similarly, a human but not a bovine a transgene was expressed in placenta in transgenic mice. Thus, placenta-specific expression of the human a-subunit gene may be the consequence of the recent evolution of a functional CRE. Expression of the human a transgene in mouse placenta further suggests that evolution of placenta-specific trans-acting factors preceded the appearance of this element. Finally, in contrast to their divergent patterns of placental expression, both the human and bovine a-subunit transgenes were expressed in mouse pituitary, indicating differences in the composition of the enhancers required for pituitary-and placenta-specific expression.The glycoprotein hormone family consists of luteinizing hormone, follicle-stimulating hormone, thyroid-stimulating hormone, and chorionic gonadotropin. These hormones control diverse biological functions, including sexual function, pregnancy, and metabolism. They are structurally similar, each consisting of an a subunit, common to all four hormones, and a unique, noncovalently associated ,B subunit (27). Expression of the glycoprotein hormone genes is controlled by a number of hormones, including sex steroids (21, 23, 26), thyroid hormone (30), hypothalamic hormones (15,16,25), and intracellular signals that act through cyclic AMP (cAMP)-dependent protein kinase A and protein kinase C (1, 6-8, 31). The genes are also under strict tissue-specific control. All mammals synthesize luteinizing, follicle-stimulating, and thyroid-stimulating hormones in the pituitary, whereas synthesis of chorionic gonadotropin occurs in the placentas of only primates and horses (...