This study aims at investigating and characterizing GnRH gene expression and regulation in human placental cytotrophoblasts by estradiol. RT-PCR analysis demonstrates that the cytotrophoblasts expressed the pro-GnRH gene. PCR with two pairs of oligonucleotide primers generated doublets 870 bases apart at the expected molecular weights, indicating that the upstream and downstream transcriptional start sites of the GnRH gene were utilized and that transcripts with and without intron I were both produced. In human placental cytotrophoblasts, the upstream transcription start site of GnRH gene was the major one and gave rise to an mRNA level three times higher than the downstream start site. RT-PCR assay also revealed that estradiol downregulated the expression of GnRH gene in human cytotrophoblasts. Estradiol at the concentration of 10nM led to a significant (50%) reduction in the mRNA from the upstream transcription start site. Such downregulation was dose-dependent and abolished by the addition of an estrogen antagonist, ICI, indicating that the gene expression was mediated by estrogen receptors. Furthermore, downregulation of GnRH gene expression by estradiol has also been confirmed by luciferase assay.