Polymorphonuclear neutrophils contain at least four serine endopeptidases, namely neutrophil elastase (NE), proteinase 3 (PR3), cathepsin G (CatG), and NSP4, which contribute to the regulation of infection and of inflammatory processes. In physiological conditions, endogenous inhibitors including a2-macroglobulin (a2-M), serpins [a1-proteinase inhibitor (a1-PI)], monocyte neutrophil elastase inhibitor (MNEI), a1-antichymotrypsin, and locally produced chelonianins (elafin, SLPI) control excessive proteolytic activity of neutrophilic serine proteinases. In contrast to human NE (hNE), hPR3 is weakly inhibited by a1-PI and MNEI but not by SLPI. a2-M is a large spectrum inhibitor that traps a variety of proteinases in response to cleavage(s) in its bait region. We report here that a2-M was more rapidly processed by hNE than hPR3 or hCatG. This was confirmed by the observation that the association between a2-M and hPR3 is governed by a kass in the ≤ 10^5 M-1s-1 range. Since a2M-trapped proteinases retain peptidase activity, we first predicted the putative cleavage sites within the a2-M bait region (residues 690–728) using kinetic and molecular modeling approaches. We then identified by mass spectrum analysis the cleavage sites of hPR3 in a synthetic peptide spanning the 39-residue bait region of a2-M (39pep-a2-M). Since the 39pep-a2-M peptide and the corresponding bait area in the whole protein do not contain sequences with a high probability of specific cleavage by hPR3 and were indeed only slowly cleaved by hPR3, it can be concluded that a2-M is a poor inhibitor of hPR3. The resistance of hPR3 to inhibition by endogenous inhibitors explains at least in part its role in tissue injury during chronic inflammatory diseases and its well-recognized function of major target autoantigen in granulomatosis with polyangiitis.