The secretory leukocyte protease inhibitor (SLPI), elafin, and its biologically active precursor trappin-2 are endogeneous low-molecular weight inhibitors of the chelonianin family that control the enzymatic activity of neutrophil serine proteases (NSPs) like elastase, proteinase 3, and cathepsin G. These inhibitors may be of therapeutic value, since unregulated NSP activities are linked to inflammatory lung diseases. However SLPI inhibits elastase and cathepsin G but not proteinase 3, while elafin targets elastase and proteinase 3 but not cathepsin G. We have used two strategies to design polyvalent inhibitors of NSPs that target all three NSPs and may be used in the aerosol-based treatment of inflammatory lung diseases. First, we fused the elafin domain with the second inhibitory domain of SLPI to produce recombinant chimeras that had the inhibitory properties of both parent molecules. Second, we generated the trappin-2 variant, trappin-2 A62L, in which the P1 residue Ala is replaced by Leu, as in the corresponding position in SLPI domain 2. The chimera inhibitors and trappin-2 A62L are tight-binding inhibitors of all three NSPs with subnanomolar K(i)s, similar to those of the parent molecules for their respective target proteases. We have also shown that these molecules inhibit the neutrophil membrane-bound forms of all three NSPs. The trappin-2 A62L and elafin-SLPI chimeras, like wild-type elafin and trappin-2, can be covalently cross-linked to fibronectin or elastin by a tissue transglutaminase, while retaining their polypotent inhibition of NSPs. Therefore, the inhibitors described herein have the appropriate properties to be further evaluated as therapeutic anti-inflammatory agents.