Analysis of the human gonadotropin-releasing hormone receptor (hGnRHR) gene 5' flanking region revealed the presence of multiple TATA, CCAAT, and transcription start sites. In addition, at least three different transcripts (5.0, 2.5, and 1.5 kb) were detected by Northern blot analysis. Taken together, these data indicated the existence of multiple promoter elements in the hGnRHR gene, and these promoters are responsible for the multiplicity of regulation of human reproductive functions. In this report, by progressive 5' and 3' deletion (-2197 to -1351, relative to the ATG) and NotI linker scanning mutagenesis coupled to transient transfection into the mouse gonadotrope-derived alpha T3-1 cell, a distal promoter element was identified at -1705/-1674. The promoter was located immediately 5' to a previously identified CAP site at -1673 in human pituitary and it drove a 17.6-+/- 1.0-fold increase in reporter gene activity. Within the promoter, a pyrimidine-rich initiator element (Inr) (-1682) and a CCAAT box (-1702) were found and mutation of these elements abrogated both protein bindings and promoter activities. By 1- and 2-D South-Western blot assays, multiple nuclear factors (40 to 54 kDa) were found to interact specifically with this promoter element. These nuclear factors were also present in other cells, including COS-7, JEG-3, and SKOV-3 cells, and these findings were consistent with functional studies which showed that the promoter is also active in these cells.