Mapping of the tryptase locus on chromosome 17 revealed a novel gene 2.3 kilobase 3' of the mouse mast cell protease (mMCP) 6 gene. This 3.7-kilobase gene encodes the first example of a protease in the tryptase family that contains a membrane-spanning segment located at its COOH terminus. Comparative structural studies indicated that the putative transmembrane tryptase (TMT) possesses a unique substrate-binding cleft. As assessed by RNA blot analyses, mTMT is expressed in mice in both strain- and tissue-dependent manners. Thus, different transcriptional and/or post-transcriptional mechanisms are used to control the expression of mTMT in vivo. Analysis of the corresponding tryptase locus in the human genome resulted in the isolation and characterization of the hTMT gene. The hTMT transcript is expressed in numerous tissues and is also translated. Analysis of the tryptase family of genes in mice and humans now indicates that a primordial serine protease gene duplicated early and often during the evolution of mammals to generate a panel of homologous tryptases in each species that differ in their tissue expression, substrate specificities, and physical properties.