Sequence analyses have revealed the existence of homology between certain aeroallergens and proteolytic enzymes. This homology can be expressed functionally, but its significance to airway pathophysiology is unknown. Studies with Madin-Darby canine kidney cells and canine tracheal epithelial cells grown on plastic substrata or matrix proteins suggest that Der p1, a major allergen from the house dust mite Dermatophagoides pteronyssinus and a cysteine proteinase, or the unfractionated growth medium extract (SGME) from which it was purified, are both capable of causing cell detachment. The ability of both agents to produce functional changes in the barrier function of the epithelium was further demonstrated using isolated bovine airway preparations. Over a 3-h duration, both Der p1 and SGME elicited significant increases in the permeability of isolated sheets of bronchial mucosa to serum albumin. Exposure of isolated bronchial segments to luminally applied solutions of Der p1 resulted in histologic evidence of epithelial injury. Neither Der p1 nor SGME was active in these experimental systems unless chemically reduced, suggesting that the effect was initiated by cysteine proteinase activity. Similar augmentation of mucosal permeability and tissue injury was produced by bovine trypsin and collagenase from Clostridium histolyticum. In both the isolated mucosal sheet model and in cultured cells, the actions of Der p1 or SGME were associated with relatively little cytolysis, suggesting a specific action of the reagents on cell attachment. These results demonstrate a new functional activity of Der p1 that may be germane to the processes of allergen presentation, inflammatory cell activation, and chronic tissue injury.