Factor XI is the zymogen of a plasma protease produced primarily in liver that is required for normal blood coagulation. We cloned approximately 2600 base pairs of the human factor XI gene upstream of exon one, identified transcription start sites, and conducted a functional analysis. Luciferase reporter assays demonstrate that the 381 base pairs upstream of exon one are sufficient for maximum promoter activity in HepG2 hepatocellular carcinoma cells. The removal of 19 base pairs between -381 and -363 results in a nearly complete loss of promoter activity. This region contains the sequence ACTTTG, a motif required for binding of the transcription factor hepatocyte nuclear factor 4alpha (HNF-4alpha) to the promoters of several genes. Gel mobility shift assays using HepG2 or rat hepatocyte nuclear extract confirm HNF-4alpha binds between bp -375 and -360. Scrambling the ACTTTG motif completely abolishes promoter activity in luciferase assays. The factor XI promoter functions poorly when transfected into HeLa carcinoma cells, and gel mobility shift experiments with HeLa nuclear extracts demonstrate no HNF-4alpha binding to the ACTTTG sequence. When a rat HNF-4alpha expression construct is co-transfected into HeLa cells, factor XI promoter activity is enhanced approximately 10-fold. We conclude that HNF-4alpha is required for hepatocyte-specific expression of factor XI.