Previously, we demonstrated that proteolytic processing within the globular domain of the α3 subunit of laminin-5 (LN5) converts LN5 from a cell motility-inducing factor to a protein complex that can trigger the formation of hemidesmosomes, certain cell-matrix attachment sites found in epithelial cells. We have prepared a monoclonal antibody (12C4) whose epitope is located toward the carboxy terminus of the globular domain of the α3 laminin subunit. This epitope is lost from the α3 subunit as a consequence of proteolytic processing. Antibody 12C4 stains throughout the matrix of cells that fail to process the α3 laminin subunit, but does not recognize the matrix of confluent cultures of MCF-10A cells, which efficiently process their α3 laminin chain. In subconfluent populations of MCF-10A cells, 12C4 only stains matrix deposited at the outer edges of cell colonies. In these cells, integrin α3β1 occasionally colocalizes with the staining generated by the 12C4 antibody but α6β4 integrin does not. In wounded MCF-10A cell cultures, the 12C4 antibody stains the extracellular matrix beneath those cells at the very edge of the cellular sheet that moves to cover the wound site. A similar phenomenon is observed in human skin wounds, since we also detect expression of the unprocessed α3 laminin subunit at the leading tip of the sheet of epidermal cells that epithelializes skin wounds in vivo. In addition, using α3 laminin subunit and integrin function-inhibiting antibodies, we provide evidence that LN5 and its two integrin receptors (α6β4 and α3β1) appear necessary for wound healing to occur in MCF-10A cell culture wounds. We propose a model for healing of wounded epithelial tissues based on these results.