The serine protease complement factor B (Bf), an acute phase plasma protein, is a component of the alternative pathway of complement activation. Previous studies revealed that several cytokines including IFN-gamma and IL-1 are involved in mediating acute phase Bf expression. To determine the molecular details of Bf expression we isolated, sequenced and characterized the 5' flanking regions of the human and murine Bf genes. In both species the Bf transcriptional start site in liver was located less than 400 bp 3' to the polyadenylation site of the upstream C2 gene. This upstream intergenic region contained greater than 65% nucleotide homology between species. Within this region, an IRS and three heat shock consensus elements were found in the murine sequence in an identical position to that of the human. To examine the functional details of Bf expression, a series of mouse and human Bf promoter-chloramphenicol acetyltransferase (CAT) chimeric gene constructs were transfected into mouse L or human HepG2 cells. Analysis of expression of these fusion gene constructs revealed that 1) cis-acting DNA sequences identified, at least in part, in the 3' untranslated region of the C2 gene (within the 400 bp upstream of the Bf cap site) mediate responsiveness to IL-1 and IFN-gamma, 2) the responsiveness to each mediator appears to be conferred by separate upstream regions similar in position and homologous in man and mouse, and 3) the IL-1 responsive region in both species appears to have the characteristics of an enhancer element. The results of this analysis suggest a selective pressure to conserve the intergenic sequence between C2 and Bf genes and that further studies of these sequences will be useful in elucidating mechanisms controlling the acute phase response.