The enhancer of the rat ornithine transcarbamylase gene is located 11 kilobases upstream from the transcription start site and has been shown to be hepatoma cell-specific. Using transgenic mice, we showed that this enhancer is capable of activating transcription in a liver-specific manner, inverting the tissue specificity of the homologous promoter that is by itself more active in the small intestine than in the liver. Transient transfection analysis with cultured hepatoma cells indicated that the enhancer activity resides in the approximately 110-base pair region containing four protein-binding sites, two for hepatocyte nuclear factor-4 (HNF-4) and two for CCAAT/enhancer binding protein (C/EBP), both of which are liver-selective transcription factors. Concatemerization of a region containing one HNF-4 and one C/EBP site led to reconstitution of the hepatoma cell-specific enhancer, and intactness of these two sites was strictly required for the enhancer activity. Furthermore, cotransfection experiments showed that both HNF-4 and C/EBP beta are necessary, and neither alone sufficient, for activation of the reconstituted enhancer in nonhepatic cells. Requirement of combinatorial operation of at least two liver-enriched transcription factors for transcriptional activation successfully explains why these liver-selective but not strictly liver-specific factors can confer more restricted liver specificity on transcription of their target genes.