The expression of selected metalloproteinases and tissue inhibitors of metalloproteinases (TIMP) was examined in three squamous cell carcinoma (SCC) cell lines (FaDu, SiHa, A431) and a keratinocyte cell line (HaCaT) to determine which metalloproteinases function in SCC invasiveness. A Matrigel invasion assay was used to assess invasiveness of the cell lines. Only the FaDu cell line showed invasiveness in this assay, and invasion of Matrigel by FaDu cells was inhibited by treatment with the metalloproteinase inhibitor, batimastat. No correlation was found between mRNA expression for matrilysin, stromelysins 1-3, TIMP-1, or TIMP-3 and secretion of these proteins, indicating that the extracellular activity of these molecules is regulated post-transcriptionally. The SCC cell lines differed from the HaCaT line in that matrilysin and TIMP-1 proteins were detected in conditioned medium from all SCC cell lines, but not in medium from HaCaT cells. Only the invasive cell line, FaDu, released active stromelysin-1 into the culture medium. These results indicate that while matrilysin contributes to the invasive phenotype, activation of stromelysin-1 is a key regulatory step for invasiveness in SCC cells.