Tryptases α and β are trypsin-like serine proteinases expressed in large amounts by mast cells. β-Tryptase is a tetramer that has enzymatic activity, but requires heparin binding to maintain functional and structural stability, whereas α-tryptase has little, if any, enzymatic activity but is a stable tetramer in the absence of heparin. As shown previously, these differences can be mainly attributed to the different conformations of the 214–220 segment. Interestingly, the replacement of Asp216 by Gly, which is present in β-tryptase, results in enzymatically active but less stable α-tryptase mutants. We have solved the crystal structures of both the single (D216G) and the double (K192Q/D216G) mutant forms of recombinant human αI-tryptase in complex with the peptide inhibitor leupeptin, as well as the structure of the non-inhibited single mutant. The inhibited mutants exhibited an open functional substrate binding site, while in the absence of an inhibitor, the open (β-tryptase-like) and the closed (α-tryptase-like) conformations were present simultaneously. This shows that both forms are in a two-state equilibrium, which is influenced by the residues in the vicinity of the active site and by inhibitor/substrate binding. Novel insights regarding the observed stability differences as well as a potential proteolytic activity of wild-type α-tryptase, which may possess a cryptic active site, are discussed.