Mouse mast cells differentially express at least four chymases (mouse mast cell protease (mMCP) 1, mMCP-2, mMCP-4, and mMCP-5), a tryptase (mMCP-6), and an exopeptidase (mouse mast cell carboxypeptidase A (mMC-CPA)). The previously uncharacterized 2.5-kilobase mMCP-2 gene was isolated and found to consist of 5 exons. The 5'-flanking region of this gene is 89, 93, and 42% similar to that of the mMCP-1, mMCP-4, and mMCP-5 genes, respectively. Inheritance patterns of restriction-enzyme fragment length polymorphisms of these six mast cell protease genes in recombinant inbred mouse strains and interspecific backcrosses were used to determine their chromosomal locations. The mMCP-6 and mMC-CPA genes are located on chromosomes 17 and 3, respectively, whereas the four mast cell chymase genes all reside on chromosome 14 linked to a gene complex that encodes four cytotoxic T lymphocyte granzymes. Pulsed-field gel electrophoresis of genomic DNA digests demonstrated that the mMCP-1, mMCP-2, and mMCP-5 genes are within 850 kilobases of each other. Although clustering of the serine protease genes on chromosome 14 may be important at a higher level of genomic organization, the ability to independently induce or suppress the steady-state levels of the four chymase transcripts by treatment of mast cells with cytokines suggests that gene clustering is not the most critical factor for coordinate expression of these proteases. Because of the unique features of their tertiary structures, the substrate specificities of the serine proteases encoded by genes at the chromosome 14 complex are predicted to be more limited than those of pancreatic chymotrypsin and pancreatic trypsin, whose genes reside on chromosomes 8 and 6, respectively. Based on present day genomic distribution and sequence similarities, we propose that a primordial gene that encoded a serine protease with restricted substrate specificity underwent extensive duplication and divergence to form a family of cytokine-regulated transcripts from genes on chromosome 14.