Previous studies suggested that GnRH gene transcripts in human tissues may be derived from an upstream transcriptional start site in addition to the well characterized hypothalamic start site. To resolve this issue we characterized the transcriptional start sites of the human GnRH gene in a human placental tumor cell line (JEG) and a human breast tumor cell line (MDA). Using primer extension and reverse transcription-polymerase chain reaction (RT-PCR) assay, we identified a discrete upstream transcriptional start site 579 bases up-stream from the hypothalamic site in both JEG and MDA cell lines. The up-stream start site lacks the TATA and CAAT elements often present in RNA polymerase-II promoters, but contains the sequence GGTCTTGCT located 84 bases 5' to the up-stream start site similar to other genes that lack TATA/CAAT boxes. RT-PCR quantitation shows that the up-stream start site is the major transcriptional start site, representing 74% of the cytoplasmic transcripts in JEG cells and 67% in MDA cells. Supporting this observation, transfection assay using a human GnRH promoter/luciferase reporter gene construct containing only the up-stream transcription start site has a higher level of transcriptional activity than the human GnRH promoter/luciferase reporter construct containing only the down-stream start site. A high relative abundance (approximately 45%) of total GnRH mRNAs were also found in the nucleus of both cell lines, which did not appear to be a consequence of the nuclear/cytoplasmic fractionation procedure. To determine if this upstream start site was used in normal GnRH-expressing human tissues, we analyzed RNA from a variety of postmortem/surgical procedure tissue samples. RT-PCR analysis together with Southern blot analysis demonstrated the presence of GnRH mRNA in human pituitary, cerebral cortex, testes, ovary, and mammary gland for the first time as well as verified GnRH gene expression in hypothalamus and placenta. The up-stream transcriptional start site is used only in reproductive tissues, such as placenta, testes, ovary, and mammary gland, suggesting tissue-specific regulation at this site.