The present study was undertaken to determine the role of the metalloproteinase MMP-9 in the invasive phenotype of squamous-cell carcinoma of the oral cavity and the regulation of its expression. Zymographic analysis of conditioned medium from 2 highly invasive squamous-cell-carcinoma cell lines indicated large amounts of an enzyme which was indistinguishable, in size (92 kDa) from the MMP-9 pro-enzyme. Conversion of the 92-kDa gelatinase into a lower-molecular-weight species (84 kDa), identical in size to the activated gelatinase, was evident when both cell lines, which are avid secretors of urokinase, were cultured in the presence of plasminogen. Penetration of an extracellular-matrix-coated filter was dramatically reduced in the presence of the collagenase inhibitor, tissue inhibitor of metalloproteinase-2, suggesting a critical role for MMP-9 in the invasive process. Immunohistochemical studies demonstrating the presence of MMP-9 in tumor cells of resected squamous-cell cancers suggested that secretion of this collagenase by cells in vitro was reflective of the in vivo setting. Since several phorbol-ester response elements are present in the MMP-9 promoter, we determined the role of protein-kinase-C pathways in the regulation of MMP-9 expression in cultured SCC. Treatment of cells with PMA resulted in a more-than-20-fold increase in the level of protein and mRNA. Conversely, culturing of cells in the presence of the protein-kinase-C inhibitor, calphostin-C, led to a dose-dependent decrease in the amount of MMP-9 mRNA and protein, suggesting that the constitutive expression of this collagenase reflects activation of this signal transduction pathway. In summary, our data suggest that, for a sub-population of squamous-cell carcinomas, secreted MMP-9 is an important determinant of the invasive phenotype, and that the expression of this metalloproteinase is regulated by protein-kinase-C pathways.