Remodeling of the extracellular matrix during tissue development, wound repair and tumor cell invasion depends on the coordinated regulation of cell adhesion receptors, matrix proteins and enzymes that proteolyse the extracellular matrix. Integrin α3β1 is a major receptor on epidermal keratinocytes for laminin-5 in the cutaneous basement membrane and is required for normal basement membrane organization during skin development. α3β1 is also expressed at high levels in the majority of adherent transformed cells and in most tumors, and it could have similar roles in extracellular matrix remodeling during tumorigenesis and cell invasion. In the present study, we show that α3β1 expression is required in immortalized mouse keratinocytes (MK) for the production of the matrix metalloproteinase MMP-9/gelatinase B, an MMP that is coexpressed with α3β1 in epithelial cell carcinomas and during wound healing, and contributes to the invasive potential of some tumor cells. MMP-9 was expressed in MK cells derived from wild-type mice, but not in MK cells derived from α3-null mice. Reconstitution of α3β1 expression in α3-null MK cells through transfection with the α3 subunit restored MMP-9 secretion, indicating an α3β1-dependent pathway for MMP-9 production. α3β1- dependent expression of MMP-9 was associated with the immortalized phenotype, since nonimmortalized, primary keratinocytes required soluble growth factors, but not α3β1, for efficient expression of MMP-9. Our results suggest that an α3β1-independent pathway(s) for MMP-9 production is suppressed in keratinocytes immortalized with large T antigen, and that an α3β1-dependent pathway is required for sustained production of MMP-9 in the absence of other pathways.