Trypsin-related serine proteases are encoded by a very large gene family in mammals. We describe here a comparative analysis of the genomic DNA sequences of mouse, rat, and human mast-cell-specific serine protease genes. Strong evidence was found for multiple exchanges of genetic information between closely related members of this gene family. The 5' regulatory regions of MMCP-1 and MMCP-L share a remarkably high degree of sequence identity (98%), starting 10 base pairs downstream of exon 1 and extending to the end of the presently sequenced region at position -1347 of the MMCP-1 gene. The remaining parts of the two genes share approximately 80% sequence identity. Evidence for at least two additional, but not so recent, exchanges was found in the 3' regions of the MMCP-4 and MMCP-L genes and in the 5' regions of the genes for MMCP-1 and MMCP-2. The 5' regulatory regions of all presently characterized mouse mast-cell-specific chymotrypsin-like serine protease genes exhibit over 88% sequence identity in the region from the transcription initiation site to approximately position -600. An exception is MMCP-5 which is the most distantly related member of this subfamily. The high degree of sequence similarities indicates a strong evolutionary homogenization of the 5' regulatory region, possibly by several gene conversion events. In addition, several insertions of genetic information have been identified in genes for mast-cell chymases and genes for T-cell granzymes. A number of these have been found to represent repetitive sequences, such as L1. The previously characterized tissue-specific enhancer element of the RMCP II gene was identified as a member of a middle repetitive sequence. A cDNA for a newly discovered pseudogene, closely related to the mouse mast cell chymases was isolated by polymerase chain reaction amplification from a mouse connective tissue-like mast cell line. The structure of this cDNA is presented. We also present the characterization of a novel spliced variant of MMCP-6 that contains an alternative 3' terminal exon (exon 6). The function of this variant, if any, is still unknown. A comparative analysis of amino acid sequence identities between different hematopoietic serine proteases shows that a high degree of sequence similarity does not always correlate with relateness in cleavage specificity. This indicates that the substrate specificity evolved with a higher evolutionary rate than the degree of overall amino acid sequence identity of these proteases.