The human airway trypsin-like protease (HAT) is a respiratory epithelium-associated, type II transmembrane serine-protease, which is also detected as an extracellular enzyme in lung fluids during airway inflammatory disorders. We have evaluated its capacity to affect the urokinase receptor (uPAR), a membrane glycolipid-anchored, three-domain (D1D2D3) glycoprotein playing crucial roles in innate immunity and inflammation by supporting cell migration and matrix degradation, and which structure and biological properties can be regulated via limited endoproteolysis. Through immunoblotting, flow immunocytometry and ELISA analyses applied to a recombinant uPAR protein, and to uPAR-expressing monocytic and human bronchial epithelial cells, it was shown that exposure of uPAR to soluble HAT in the range 10 to 500 nM resulted in the proteolytic processing of the full-length (D1D2D3) into a truncated (D2D3) species, cleavage occurring in the D1 to D2 linker sequence after Arg residues at position 83 and 89. Using immunohistochemistry, HAT and uPAR were both found to be expressed in the human bronchial epithelium. Moreover, transient co-transfection in epithelial cells showed that membrane co-expression of the two partners produced a constitutive and extensive shedding of the D1 domain, occurring for membrane-associated HAT concentrations in the nanomolar range. Because the truncated receptor was found to be unable to bind two of the major uPAR ligands, the adhesive matrix protein vitronectin and the serine-protease urokinase, it thus appears that proteolytic regulation of uPAR by HAT is likely to modulate cell adherence and motility, as well as tissue remodeling during the inflammatory response in the airways.