Factor VII is a vitamin K-dependent coagulation protein essential for proper hemostasis. The human Factor VII gene spans 13 kilobase pairs and is located on chromosome 13 just 2.8 kilobase pairs 5 to the Factor X gene. In this report, we show that Factor VII transcripts are restricted to the liver and that steady state levels of mRNA are much lower than those of Factor X. The major transcription start site is mapped at ؊51 by RNase protection assay and primer extension experiments. The first 185 base pairs 5 of the translation start site are sufficient to confer maximal promoter activity in HepG2 cells. Protein binding sites are identified at nucleotides ؊51 to ؊32, ؊63 to ؊58, ؊108 to ؊84, and ؊233 to ؊215 by DNase I footprint analysis and gel mobility shift assays. A liver-enriched transcription factor, hepatocyte nuclear factor-4 (HNF-4), and a ubiquitous transcription factor, Sp1, are shown to bind within the first 108 base pairs of the promoter region at nucleotide sequences ACTTTG and CCCCTCCCCC, respectively. The importance of these binding sites in promoter activity is demonstrated through independent functional mutagenesis experiments, which show dramatically reduced promoter activity. Transactivation studies with an HNF-4 expression plasmid in HeLa cells also demonstrate the importance of HNF-4 in promoting transcription in nonhepatocyte derived cells. Additionally, the sequence of a naturally occurring allele containing a previously described decanucleotide insert polymorphism at ؊323 is shown to reduce promoter activity by 33% compared with the more common allelic sequence.