A rhesus monkey genomic library was screened with a cDNA for the glycoprotein hormone alpha-subunit. Genomic clones hybridizing with exon-specific probes were selected and the DNA sequences were determined for 1.6 kb of 5'-flanking DNA, all four exons, the second and third introns, all exon-intron junctions, and 357 bp of 3'-flanking DNA. Comparison with the 236 bp of 5'-flanking sequence data available for the human alpha gene indicates an overall homology of 95%. Primer extension analysis of rhesus placental and pituitary mRNA demonstrated that transcription initiation is identical to that in the human placenta. The rhesus gene contains an element nearly identical (21/22 bases) to the placental tissue-specific element described for the human alpha gene. The rhesus gene has only one copy of the cAMP-response element (CRE), which is present as a direct repeat in the human gene. The rhesus CRE contains the consensus core sequence TGACG-TCA with the cytosine in the fourth position that is essential for placental expression of the human gene. The 5'-flanking region also has elements highly homologous to the consensus estrogen and progesterone/glucocorticoid response elements, as well as thyrotrope-specific and Pit-1-like binding sites described in rodent genes. The nucleotide sequence of four exons (predicted mRNA) have an aggregate homology of 92.7% with the human sequence. However, a 12-bp insertion to the second exon results in the addition of 4 amino acids to the amino-terminal end of the protein; these are homologous with the proteins of nonprimates but are lacking in the human alpha-subunit. The amino acid sequence of the deduced protein was slightly more homologous with the bovine than the human protein (91.6% vs. 89.6%). Thus, the rhesus glycoprotein alpha-subunit gene codes for a protein whose structure somewhat more closely resembles that of lower species, but the 5'-flanking DNA of the gene has gained the elements necessary for transcription in the placental syncytiotrophoblast which distinguishes the primate placenta from the other species examined.