Ghrelin is a recently discovered stomach hormone and endogenous ligand for the GH secretagogue receptor. The aim of these studies is to elucidate molecular mechanisms underlying regulation of the ghrelin gene. Distal and proximal transcription initiation sites are present. A short transcript, a product of the proximal site, showed a more widespread distribution. Two sets of 5'-upstream segments of the rat and human ghrelin genes were cloned and sequenced. Rat promoter segments upstream of the distal site showed highest activity in kidney (COS-7) and stomach (AGS) cells, whereas human promoter segments upstream of the proximal site showed highest activity in AGS and pituitary (GH3) cells in transient transfection assays. For the human, the core promoter spanned -667 to -468 bp, including the noncoding exon 1 and a short 5' sequence of intron 1. For the rat, the core promoter spanned -581 to -469 bp, and inclusion of exon 1 and a short 5'-sequence of intron 1 reduced activity by 67%. Mutation of initiator-like elements in the rat lowered activity by 20-50%, whereas in the human, all activity was abolished. Overexpression of upstream stimulatory factors increased ghrelin core promoter activity. Fasting increases stomach ghrelin expression, glucagon-a fasting-induced hormone, increased ghrelin expression in vivo in rats, and promoter activity by approximately 25-50%. Together, these findings indicate that structural differences between the rat and human ghrelin core promoters may account in part for the differences in their transcriptional regulation. Nonetheless, upstream stimulatory factor and glucagon exert similar effects on regulation of rat and human ghrelin promoters.