Elafin was shown to be a new type of proteinase inhibitor which has an anchoring sequence. Human elafin, a potent inhibitor specific for elastase and proteinase 3, has a unique repeating sequence in its prosegment that is rich in Gin and Lys residues. The prosegment, termed “cementoin,” exhibits high homology with the repetitive element of seminal vesicle clotting protein, which is known as a good substrate for prostate transglutaminase. The cross-linking of cementoin by tissue transglutaminase showed that the cementoin moiety is indeed a preferable substrate for transglutaminase. In addition, transglutaminase-mediated cross-linking between cementoin and laminin was observed in vitro, suggesting that cementoin has the ability to covalently attach to other extracellular matrix proteins. To determine whether or not this type of covalent gluing of elafin through the cementoin moiety occurs in vivo, we determined the molecular size of cementoin-elafin in the trachea mucous epithelium by Western blotting; the rationale of this approach is that (i) the trachea is the richest source of cementoin-elafin, as shown below, and (ii) if cementoin-elafin is covalently associated with other proteins, it should migrate as a higher M_r species on SDS-polyacrylamide gel electrophoresis; cementoin-elafin immunoreactivity was indeed detected at a position corresponding to 50 kDa, a value much higher than that of its monomeric form. RNase protection analysis and immunohistochemical staining revealed that cementoin-elafin is densely distributed in the skin and trachea, and moderately in the stomach, duodenum and small intestine. These sites of localization are consistent with the locations where elastic fibers are abundant. These findings suggest that the cementoin moiety serves as molecular glue for anchoring elafin to extracellular matrix proteins in order to protect elastic tissues. Elafin may therefore represent a new class of proteinase inhibitors.