The novel tetrameric structure of human β-tryptase faces each active site into the central pore, thereby restricting access of most biologic protease inhibitors. The mechanism by which the anti-tryptase mAb B12 inhibits human β-tryptase peptidase and proteolytic activities at neutral pH, but augments proteolytic activity at acidic pH, was examined. At neutral pH, B12-β-tryptase complexes are inactive. At acidic pH, B12 (intact and Fab) minimally affects peptidase activity when added to β-tryptase tetramers, but does induce susceptibility to inhibition by soybean trypsin inhibitor and antithrombin III. Surprisingly, B12 Fab-β-tryptase complexes formed at both neutral and acidic pH exhibit the apparent molecular mass of a complex with 1 β-tryptase monomer and 1 Fab by gel filtration. B12 does not compete with heparin for binding to tryptase at either neutral or acidic pH. Thus, B12 directly disrupts β-tryptase tetramers to monomers that are inactive at neutral pH, whereas at acidic pH, are active and more accessible to protein inhibitors and substrates.