Minor cell defects in epithelial sheets are a consequence of superficial injury in natural intestinal diseases. These defects are restored by migration of shouldering epithelial cells over the underlying matrix. Restitution of minor epithelial defects is facilitated by collagen IV (R. Moore, J. Madri, S. Carlson, and J. L. Madara. Gastroenterology 102: 119-130, 1992). Here we characterize enterocyte attachment to substrates and determine the role of divalent cations. Attachment and spreading of native intestinal epithelial cells (EC) isolated by mechanical vibration were greatest on collagen IV [compared with collagen I, collagen III, laminin, fibronectin, and plastic; P < 0.001]. Attachment was not inhibited by cycloheximide but was inhibited by cytochalasin D and by a functionally defined antibody to collagen IV. In fixed extracellular 1.25 mM Mg2+, EC attachment was nearly three times greater in nominally Ca(2+)-free medium compared with 5 mM Ca2+ (P < 0.001). Conversely, in fixed 1.25 mM Ca2+, cell attachment was three times greater in the presence of 2.5 mM Mg2+ compared with Mg(2+)-deplete media (P < 0.001). Last, in the presence of the intracellular Ca2+ chelator 1,2-bis(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, EC attachment was diminished by > 50% (P < 0.001). We speculate that local tissue concentrations of divalent cations may play an important role in restitution of intestinal epithelial wounds by mediating attachment of migrating EC to collagen IV.