An elevated plasma fibrinogen level is an independent risk factor for cardiovascular disease. Therefore, an understanding of the regulation of fibrinogen expression is important. Inflammation and genetic variation of the fibrinogen beta gene regulate plasma fibrinogen levels, and there are indications that inflammation and genetic variation interact. The aim of our study was to gain more understanding of the regulation of the inflammatory response of the fibrinogen beta gene and to determine the effects of genetic variation. Luciferase reporter gene assays in hepatoma cells, mutation analysis, and electrophoretic mobility shift assays were used to investigate the transcriptional regulation of the fibrinogen beta promoter. We identified a hepatocyte nuclear factor-3 (HNF-3) site located just upstream of previously identified interleukin-6 (IL6)-responsive sequences. This HNF-3 site is essential for a full response of the promoter to IL6, which is a new function for HNF-3. The activity of the CCAAT box/enhancer-binding protein site (located 18 nucleotides downstream of the HNF-3 site and important to the IL6 response) depends on the integrity of the HNF-3 site and vice versa, explaining the necessity of HNF-3 in the IL6 response of the fibrinogen beta promoter. Furthermore, small interfering RNA to HNF-3 reduces the fibrinogen beta mRNA levels. The rare T allele of the -148C/T polymorphism, which is present between the binding sites of HNF-3 and CCAAT box/ enhancer-binding protein, interferes with this mechanism, and this polymorphism is in our assay system the only genetic determinant of IL6-induced promoter activity among six polymorphisms in the fibrinogen beta promoter. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Chemicals / CAS: fibrinogen, 9001-32-5; BBeta fibrinogen; DNA Primers; DNA-Binding Proteins; Fibrinogen, 9001-32-5; FOXA1 protein, human; FOXA2 protein, human; FOXA3 protein, human; Hepatocyte Nuclear Factor 3-alpha; Hepatocyte Nuclear Factor 3-beta, 135845-92-0; Hepatocyte Nuclear Factor 3-gamma, 135845-91-9; Interleukin-6; Luciferases, EC 1.13.12.-; Nuclear Proteins; RNA, Messenger; RNA, Small Interfering; Transcription Factors