In order to characterize the molecular mechanisms that dictate microsomal triglyceride transfer protein (MTP) gene transcription in human and hamster, two species with similar plasma lipoprotein profiles, the MTP gene promoters were cloned, sequenced, and functionally characterized by transient transfection analysis. The results presented in this report indicate that the 5' ends of human and hamster MTP genes share similar structural features. The promoter sequences are well conserved and consist of similar functional elements. Transient transfection analysis of MTP promoter-driven luciferase gene expression showed that the promoter is active in liver and intestinal cells but not in epithelial cells, consistent with endogenous MTP gene expression. The -123 to -85 bp region of the human promoter is critical for the expression and contains the consensus recognition sequences for liver cell-specific factors HNF-1 and HNF-4 and activator protein AP-1. The promoter contains a modified sterol response element and a negative insulin response element. The human promoter activity is positively regulated by cholesterol and negatively regulated by insulin. From the functional analysis of MTP promoters, it is concluded that the elements that regulate the cell type-specific expression in human and hamster are well conserved and that insulin and cholesterol can regulate the activity of the MTP promoter in opposite directions.