Although GHRH is known to play a pivotal role in the regulation of the GHRH-GH-IGF-I axis, the molecular mechanism of GHRH gene expression has not yet been examined. Here we studied the transcriptional regulation of the GHRH gene 5'promoter using an in vitro experimental model system. We especially focused on the role of homeobox transcriptional factor Gsh-1, because a dwarf phenotype and abolished GHRH expression was observed in Gsh-1 knockout mice. First, we cloned human Gsh-1, which showed 87.3% homology with mouse Gsh-1 at the nucleotide level. When the 5'-promoter region of the rat GHRH gene was introduced into the human placental cell line JEG-3, in which we found the endogenous expression of Gsh-1 as well as GHRH mRNA, substantial transcriptional activity of the promoter was recognized. Promoter activity was further enhanced by overexpression of Gsh-1 protein, whereas it was substantially reduced by elimination of Gsh-1 binding sites. EMSA confirmed the actual binding of Gsh-1 on the multiple binding sites of GHRH gene promoter. Finally, coexpression of CREB-binding protein significantly enhanced the Gsh-1-induced GHRH gene expression, suggesting the cooperative role of the coactivator protein. Because Gsh-1 is found to be expressed in the hypothalamus of the adult rat, our data provide evidence that the Gsh-1 homeobox protein plays a key role in the expression of the GHRH gene.