Epidemiological evidence suggests that the earlier in life a woman becomes pregnant, the lower her chances for developing breast cancer later in life. This protective effect appears to be due to pregnancy hormone hCG inducing the nonreversible differentiation of proliferative terminal end buds into secretory type lobules. Perhaps this and other actions of hCG are mediated by newly discovered hCG/LH receptors in human breast cells. Thus the hCG actions in breast are potentially important for breast cancer prevention. Because of this importance, we investigated the cis-acting elements and trans-acting proteins that determine the transcription of human chorionic gonadotropin (hCG)/luteinizing hormone (LH) receptor gene in MCF-7, MDA-MB-231, and normal human breast epithelial HMEC cells. These cells contained major-4.8 and 1.8 kb-and minor-9.0, 6.0, and 1.2 kb-hCG/LH receptor transcripts with significantly higher levels in MCF-7 cells. Nuclear run-on transcription, as well as transfection with a fusion construct of luciferase gene and the -2056 to -1 bp of the 5'-flanking region of hCG/LH receptor gene, revealed that MCF-7 cells were transcriptionally more active than the other breast cells. Sequential deletion of the 5'-flanking region revealed that breast cells contained a promoter at -184 to -1 bp. Electrophoretic gel mobility shift assays demonstrated that breast cell nuclear extracts contained Ap2 and Egr promoter binding proteins. Sp1 was also present, but it could not bind because of competition with Egr for binding to a partially overlapping Egr/Sp1 site. The higher levels of Ap2 and Egr binding proteins may explain higher transcription of hCG/LH receptor gene in MCF-7 cells than in the other breast cells.