The epidermal growth factor receptor (EGFR) promotes intestinal epithelial restitution, an important early process in the re-epithelialization of ulcers. During epithelial restitution, the mechanism of EGFR activation is not known. We evaluated the role of tumor necrosis factor converting enzyme (TACE), a metalloprotease disintegrin that proteolytically processes plasma membrane-anchored EGFR ligand precursors into their mature active forms, in wound-induced EGFR activation and epithelial restitution. Using scrape-wounded RIE-1 intestinal epithelial cell monolayers to model epithelial ulceration and restitution, we observed the rapid wound-dependent release of EGFR ligands into culture medium. RIE-1 cells express TACE, and treatment with phorbol ester, an established TACE stimulus, triggered the extracellular release of an EGFR ligand, transforming growth factor-. Blockade of TACE using TAPI-1, a specific hydroxamate inhibitor of metalloprotease disintegrins, prevented release of EGFR ligands from wounded RIE-1 cell monolayers. The restitution of wounded RIE-1 cell monolayers was also dose-dependently inhibited by TAPI-1, establishing the role of metalloprotease disintegrins in this process. These results have established a mechanism of EGFR activation in wounded intestinal epithelium and show an important functional role for metalloprotease disintegrin-mediated ectodomain shedding during intestinal epithelial restitution. Therefore, activation of the TACE-EGFR system might promote the healing of intestinal tract ulcers in patients.