GHRH plays a pivotal role in the regulation of both synthesis and secretion of GH in the anterior pituitary. In this study, we examined the molecular mechanism of depolarization-induced GHRH gene transcription using the hypothalamus cell line, Gsh+/+, revealing the involvement of the transcription factor called nuclear factor of activated T cells (NFAT). GHRH, NFAT1, NFAT4, and related genes were endogenously expressed in Gsh+/+ cells and the rat arcuate nucleus, where NFAT1 and GHRH were colocalized. Cellular excitation with high potassium potently stimulated endogenous GHRH gene 5'-promoter activity as well as the NFAT-mediated gene transcription, the former being further enhanced by coexpression of NFAT. On the other hand, cyclosporin A (a calcineurin-NFAT inhibitor) or EGTA (a calcium chelator) significantly blocked the depolarization-induced GHRH gene transcription. EMSA and site-directed mutagenesis experiments showed the direct binding of NFAT at five sites of the GHRH promoter, among which the relative importance of three distal sites (-417/-403, -402/-387, -317/-301) was suggested. Finally, elimination of all five sites completely abolished the NFAT-induced GHRH gene up-regulation. Altogether, our results suggest that the transcription factor NFAT is involved in the depolarization-induced transcriptional activation of GHRH gene in the neuronal cells.