Factor B (Bf), an enzyme of the alternative pathway of complement activation, is one of four major histocompatibility complex (MHC) class III genes. To ascertain the genetic mechanism for tissue-specific constitutive and regulated expression of Bf, we sequenced the regulatory regions 5' of the gene from mice of different H-2 MHC haplotypes and assessed trans-acting factors, specific DNA binding nucleoproteins, in liver and kidney. Striking tissue-specific differences in constitutive expression of Bf were demonstrated in mice of H-2f or H-2z haplotypes when compared with H-2d or H-2u (kidney and intestinal Bf in H-2d or H-2u much greater than H-2f or H-2z). These differences correlated with a point nucleotide substitution 3 bp downstream of the upstream Bf initiation site that affects interaction with a DNA binding protein. This and additional cis differences localize the sequence substitutions responsible for previously identified restriction fragment length polymorphisms among inbred mouse strains and also reveal two previously unrecognized polymorphisms generated by SmaI and HinfI digestion. Evidence for differences in trans was found in a comparison of DNA binding nucleoproteins from kidney, but not liver, of B10.PL when compared with B10.M. These data, together with the high degree of sequence homology between human and mouse Bf 5' flanking regions, should prompt a search for polymorphic restriction sites and cis binding elements in the Bf promoter that could serve as markers of human MHC-associated renal pathology and variants in local MHC class III gene expression.