Cathepsin V, a thymus and testis-specific human cysteine protease, was expressed in Pichia pastoris, and its physicokinetic properties were determined. Recombinant procathepsin V is autocatalytically activated at acidic pH and is effectively inhibited by various cysteine protease class-specific inhibitors. The S2P2 subsite specificity of cathepsin V was found to be intermediate between those of cathepsins S and L. The substrate binding pocket, S2, accepted both aromatic and nonaromatic hydrophobic residues, whereas cathepsins L and S preferred either an aromatic or nonaromatic hydrophobic residue, respectively. In contrast to cathepsin L, but similar to cathepsin S, cathepsin V exhibited only a very weak collagenolytic activity. Furthermore, cathepsin V was determined to be significantly more stable at mildly acidic and neutral pH than cathepsin L, but distinctly less stable than cathepsin S. A homology structure model of cathepsin V revealed completely different electrostatic potentials on the molecular surface when compared with human cathepsin L. The model-based electrostatic potential of human cathepsin V was neutral to weakly positive at and in the vicinity of the active site cleft, whereas that of cathepsin L was negative over extended regions of the surface. Surprisingly, the electrostatic potential of the human cathepsin V model structure resembled that of the model structure of mouse cathepsin L. These differences in the electrostatic potential at the molecular surfaces provide a reactivity determinant that may be the source of differences in substrate selectivity and pH stability. Cathepsin V was mapped to the chromosomal region 9q22.2, a site adjacent to the cathepsin L locus. The high sequence identity and the overlapping chromosomal gene loci suggest that both proteases evolved from an ancestral cathepsin L-like precursor by gene duplication.