Precursors of α-defensin peptides require activation for bactericidal activity. In mouse smart intestine, matrilysin colocalized with α-defensins (cryptdins) in Paneth cell granules, and in vitro it cleaved the pro segment from cryptdin precursors. Matrilysin-deficient (MAT(-/-)) mice lacked mature cryptdins and accumulated precursor molecules. Intestinal peptide preparations from MAT(-/-) mice had decreased antimicrobial activity. Orally administered bacteria survived in greater numbers and were more virulent in MAT(-/-) mice than in MAT(+/+) mice. Thus, matrilysin functions in intestinal mucosal defense by regulating the activity of defensins, which maybe a common rote for this metalloproteinase in its numerous epithelial sites of expression.