The 5'-flanking sequence of the human prothrombin gene was isolated by screening a human liver phage library with a human prothrombin cDNA as a hybridization probe. A phage was identified that contained 3 kilobase pairs of DNA upstream of the initiator methionine codon. Primer extension studies showed that the major transcription initiation sites were located 23 and 36 base pairs upstream of the initiator codon. DNA sequences in the 5'-flanking region of the human prothrombin gene were then analyzed for cis-activating transcriptional activity by a transient expression system using the human growth hormone gene as the reporter gene. The chimeric expression vector was introduced into HepG2 cells, and secreted human growth hormone was monitored by using a radio-immunoassay. These studies showed that the 3-kilo-base pair fragment contained sequences that were sufficient for the initiation of transcription in HepG2 cells. Subsequent deletion studies showed that the 3-kilobase pair fragment contained two elements: a weak promoter in the region immediately upstream of the mRNA coding sequence and an enhancer located between nucleotides -860 and -940. The enhancer element was active at a distance and in either orientation. In addition, the enhancer was liver cell-specific and acted on heterologous promoters including the herpes simplex virus thymidine kinase promoter and the mouse metallothionein I promoter. Comparison of the nucleotide sequence of the enhancer with a DNA sequence data base showed the enhancer sequence to be unique. The enhancer sequence is flanked by an inverted repeat 5' CCTCCC 3' and contains a putative binding site for hepatic nuclear factor 1. Deoxyribonuclease I footprint analysis and linker scanning mutagenesis showed that the enhancer contains multiple protein binding motifs. Mutagenesis of the 3' boundary CCTCCC sequence eliminated the enhancer activity. Comparison with other liver genes showed the presence of the CCTCCC sequence in the hepatitis B virus enhancer, the alpha 1-antitrypsin promoter, and the fibrinogen beta-chain promoter, suggesting a functional role for this motif.