The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is a basic helix-loop-helix transcription factor. The hepatic expression of SHARP-2 mRNA was investigated under various conditions. The level was decreased in the regenerating rat liver and malignant hepatoma cells. In contrast, the expression of SHARP-2 mRNA was induced in rat livers by feeding a high-carbohydrate diet. To analyze the molecular mechanism involved in the regulation of the rat SHARP-2 gene expression, the gene was cloned. It was approximately 6-kb in length and consists of five exons and four introns. To investigate the transcriptional regulatory region of this gene, SHARP-2/firefly luciferase reporter plasmids were transfected into hepatoma cells. A functional analysis of 5(')-deletion constructs revealed that two E box sequences between -160 and -144 are mainly responsible for promoter activity. Although upstream stimulatory factors (USFs) bound to the element in vitro, USF2 failed to stimulate promoter activity from the element using the co-transfection experiment. Therefore, other E box-binding transcription factors differing from USF proteins or USF-associated proteins are necessary for transcriptional stimulation of the rat SHARP-2 gene.