The a and I subunit genes encoding chorionic gonadotropin (CG) are regulated transcriptionally in placental cells by cyclic AMP (cAMP). The regulatory response sequences of the a gene have been studied extensively. Similar studies of the CG j8 subunit (CGI) gene have not been possible because transcriptionally active sequences have not been identified in the clones isolated to date. The CGi subunit genes form a complex cluster of seven structurally similar genes that include six CG,(-like genes and a single luteinizing hormone J subunit (LHj8) gene. We isolated overlapping clones containing the entire CGO/LHP gene cluster (68 kilobases) from a human genomic cosmid library. The organization of the gene cluster was similar to that found in previous analyses, as determined by Southern blots of genomic DNA, but differed from some of the gene assignments, as determined by fragments cloned in A phage. The 5'-flanking sequence of the most active CGP gene (CGI5) was linked to the chloramphenicol acetyltransferase (CAT) coding sequence for analyses of transient expression in different cell types. CG(3CAT was expressed preferentially in JEG-3 choriocarcinoma cells, and expression was markedly stimulated by treatment with 8-bromo-cAMP. Deletion mutagenesis of the CGj8 5'-flanking sequence revealed that multiple regions were required for maximal expression. The kinetics for cAMP stimulation of aCAT and CGISCAT expression were different, suggesting that different pathways may be involved in cAMP-stimulated expression of the a and CGOi genes.