To identify the promoter sequence(s) of the CGβ gene, genomic fragments derived from a cosmid containing the CGβ gene family were transfected into mouse Y1 adrenal cortical cells. Using this system, we showed that the CGβ genes 5, 3, and 8 have functional promoters, the basal element of which in the case of CGβ5, was within 78 base pairs 5' ward from the CAP site. The size of the CGβ transcripts and identity of the transcription start site was the same for CGβ mRNA synthesized in Y1 cells as in first trimester placenta. The promoter region identified by this system was also capable of driving the chloramphenicol acetyltransferase gene when transfected stably into choriocarcinoma cells. Chloramphenicol acetyltransferase constructs bearing variable length of 5'-flanking sequences from CGβ5 and transfected into trophoblast cells suggest the presence of regulatory sequences within 700 base pairs from the CAP site. The information obtained here provide a foundation for studies of analyzing trans-acting placental and pituitary proteins to the defined CGβ promoter region.