The transcription factor Oct-3/4 may be important in maintaining embryonic cells in an undifferentiated state. It is probably down-regulated at about the time that human chorionic gonadotropin (hCG) is first expressed in embryonic trophectoderm. Here we report that Oct-3/4 strongly inhibits the hCGbeta subunit (hCGbeta) promoter in JAr choriocarcinoma cells. Oct-3/4 reduced chloramphenicol acetyltransferase (CAT) reporter expression from the -305hCGbeta promoter by about 90% in transient co-transfection assays, but had no effect on expression from the -249hCGbeta promoter. The -305/-249 hCGbeta fragment specifically bound synthetic Oct-3/4 protein as measured in electrophoretic mobility shift assays, and the Oct-3/4-binding site was localized around -270 by methylation interference footprinting. Site-directed mutagenesis of this binding site abolished Oct-3/4 repression. When stably transfected into JAr cells, Oct-3/4 reduced the amounts of both endogenous hCGbeta messenger RNA and hCG protein to less than 10% of controls. We suggest that silencing of Oct-3/4 in trophectoderm is a prerequisite for hCG up-regulation in early human embryos at the time of maternal recognition of pregnancy.