We have isolated, cloned, and characterized cDNA and genomic DNA corresponding to the mRNA and gene for the rat mast cell protease, RMCP II. The amino acid sequence deduced from the cDNA provides evidence that this protease is synthesized as a precursor, with a signal peptide and additional residues at the N and C termini. RNA homologous to the cDNA is expressed only in mast cells. Analysis of RNA from the two subclasses of mast cell, mucosal and serosal, indicates subclass specific expression of the different proteases found in each of these two subclasses. S1 protection analysis and the sequence of the genomic clone indicate that the serosal mast cell protease, RMCP I, is likely to be coded for by a separate, highly homologous gene. A comparison of the exon/intron structure of the RMCP II gene with genes of related serine proteases further indicates that RMCP II is a member of a family of proteases distinct from those found in the pancreas. We have also isolated a third gene, highly homologous to RMCP II but different from it and from the gene for RMCP I. Analysis of the 5' transcriptional control region of both genes showed striking homology to the TATA box and enhancer regions of the pancreatic proteases.