Entactin is the basement membrane protein which bridges laminin and type IV collagen. Entactin is known to be degraded by serine proteinases, but its susceptibility to matrix metalloproteinases has not been determined. We have studied the capacity of three matrix metalloproteinases (interstitial collagenase, 92-kDa gelatinase, and matrilysin) to degrade entactin. While all three metalloenzymes cleaved entactin, matrilysin was approximately 100-fold as effective as collagenase and 600-fold as effective as 92-kDa gelatinase. The Km of matrilysin for entactin was 8.9 x 10(-7) M. A Vmax of 21 molecules of entactin degraded/molecule of matrilysin/min at 37 degrees C was observed. An Arrhenius plot relating matrilysin's catalytic activity to temperature was linear from 15 to 37 degrees C and indicated an activation energy of 10,060 calories/mol. Matrilysin produced multiple, but distinct, cleavages in entactin resulting in peptide fragments ranging from 115 to 29 kDa. The precise sites of cleavage of six fragments were determined by Edman degradation. Cleavage sites consistently occurred amino-terminal to leucine or isoleucine. These data indicate that entactin is a substrate for matrix metalloproteinases. The effectiveness of matrilysin is noteworthy, however, particularly in relation to the minimal ability of other much more well described matrix metalloproteinases to attack this substrate. Our results suggest a potentially important role for matrilysin in disruption of basement membranes by tumor or inflammatory cells.