The blood-clotting protein fibrinogen is composed of three subunits, designated A alpha, B beta, and gamma, which are encoded by a family of related genes. As part of the acute-phase response, expression of the fibrinogen genes is coordinately regulated in the liver by glucocorticoids. To understand the factors underlying this hormonal response, we have examined control of transcription from fibrinogen gene fragments transfected into hepatocytes from the frog Xenopus laevis. This analysis is the first in any species to define transcriptional regulatory elements for the fibrinogen genes by transfection into primary liver cells, rather than liver-derived cell lines. A transfection vector was constructed containing the Xenopus B beta gene transcription start site and 1293 bp of the 5' flanking sequence linked to the firefly luciferase gene. When this construct was transfected into primary liver parenchymal cells, luciferase expression was induced approximately 14-fold by glucocorticoids, an increase similar to the transcriptional stimulation of the endogenous B beta subunit gene. DNA fragments with as little as 284 bases of upstream sequence retained full hormone responsiveness. This region contains a sequence resembling the canonical glucocorticoid response element (GRE) at bases -148 to -162. Deletions or specific point mutations eliminating this putative GRE led to complete loss of glucocorticoid inducibility. Physical association of the steroid hormone receptor with this functional GRE was demonstrated with a truncated form of the rat glucocorticoid receptor containing the DNA-binding domain. A second possible GRE at positions -526 to -540 was not hormone-responsive, in either the presence or the absence of the more proximal GRE. The regulatory region also has a sequence similar to the binding site for a liver-specific transcription factor, hepatocyte nuclear factor 1 (HNF-1), at positions -120 to -132. Specific point mutations in the HNF-1-binding site, in a construct containing a wild-type GRE, reduced promoter activity by a factor of 10, while stimulation by glucocorticoids was retained. Binding studies confirmed specific interaction between this site and the transcription factor HNF-1 alpha from mouse. Thus, we have identified a GRE sufficient to account for full glucocorticoid inducibility and an HNF-1 site close to the promoter that are major determinants of transcriptional control of the Xenopus fibrinogen B beta subunit gene in cells from normal liver tissue.