Probing of the GenBank expressed sequence tag (EST) data base with varied human tryptase cDNAs identified two truncated ESTs that subsequently were found to encode overlapping portions of a novel human serine protease (designated tryptase epsilon or protease, serine S1 family member 22 (PRSS22)). The tryptase epsilon gene resides on chromosome 16p13.3 within a 2.5-Mb complex of serine protease genes. Although at least 7 of the 14 genes in this complex encode enzymatically active proteases, only one tryptase epsilon-like gene was identified. The trachea and esophagus were found to contain the highest steady-state levels of the tryptase epsilon transcript in adult humans. Although the tryptase epsilon transcript was scarce in adult human lung, it was present in abundance in fetal lung. Thus, the tryptase epsilon gene is expressed in the airways in a developmentally regulated manner that is different from that of other human tryptase genes. At the cellular level, tryptase epsilon is a major product of normal pulmonary epithelial cells, as well as varied transformed epithelial cell lines. Enzymatically active tryptase epsilon is also constitutively secreted from these cells. The amino acid sequence of human tryptase epsilon is 38-44% identical to those of human tryptase alpha, tryptase beta I, tryptase beta II, tryptase beta III, transmembrane tryptase/tryptase gamma, marapsin, and Esp-1/testisin. Nevertheless, comparative protein structure modeling and functional studies using recombinant material revealed that tryptase epsilon has a substrate preference distinct from that of its other family members. These data indicate that the products of the chromosome 16p13.3 complex of tryptase genes evolved to carry out varied functions in humans.