The only tryptic enzymes identified so far in mouse mast cells (MCs) are three members of the chromosome 17A3.3 family of neutral proteases. Sequence analysis of a cDNA library revealed that BALB/c mouse bone marrow-derived MCs express neuropsin, a member of the chromosome 7B2 family of tryptic kallikreins. Kinetic studies revealed that neuropsin is expressed relatively early in MC development. As assessed immunohistochemically, the MCs residing in numerous connective tissues store neuropsin in their secretory granules. The finding that the neuropsin transcript is maximally expressed in the intestine at the height of a helminth infection indicates that MC-committed progenitors selectively increase their expression of neuropsin as they develop into mature mucosal MCs. This is the first report documenting the expression of neuropsin in an immune cell. Thus, it is now apparent that mouse MCs store at least two distinct families of tryptic-like proteases in their secretory granules.