Laminin 5 (alpha3beta3gamma2) is specifically present in the basal lamina underneath epithelia with secretory or protective functions, where it is essential for anchoring basal epithelial cells to the underlying extracellular matrix. Laminin 5 is produced by epithelial cells as a 480-kDa precursor that is converted into forms of 440 and 400 kDa. To analyse the processing of laminin 5, we have used monolayer and co-cultures of epithelial cells and fibroblasts. The processing of the 180-kDa laminin alpha3 chain to 165 kDa in the cell culture medium, and to both 165 and 145 kDa polypeptides in the cell layer, are not modified by the presence of fibroblasts. In contrast, cleavage of the laminin gamma2 chain, occurring in the cell culture medium and in the cell layer, is enhanced by the presence of fibroblasts. Further analysis by immunofluorescence staining and laser-scanning microscopy reveals that deposited laminin 5 is present in a fibroblast-associated filamentous meshwork. Only laminin 5 containing a fully processed gamma2 chain is present in this fibroblast-associated fraction. These studies show that, although laminin 5 is a product of epithelial cells, fibroblasts contribute to its integration into the extracellular matrix architecture.