Oral squamous cell carcinomas are highly invasive lesions that destroy adjacent tissues and invade bone and muscle, which is most likely the result of matrix metalloproteinase (MMP) activity. We examined three cell lines derived from squamous cell carcinoma of the tongue for their intrinsic capacities to degrade interstitial collagen with the goal of identifying the matrix-degrading enzymes. SCC-25 and SCC-15 cells degrade reconstituted fibrillar type I collagen in the absence of exogenous growth factors or cytokines when seeded as a colony on dried films. Degradation is confined to the subjacent matrix, is enhanced 2-3-fold by phorbol ester, and is strictly MMP-dependent, as it is blocked by BB-94 and tissue inhibitor of metalloproteinases-2 but not by inhibitors of serine and cysteine proteinases. Both cell lines express active (M(r) 57,000) membrane type I-MMP (MT1-MMP) on their surfaces, as detected by surface biotinylation and immunoprecipitation. Concomitantly, both cell lines activate endogenous MMP-2 when cultured on type I collagen films, as assessed by zymography. Phorbol ester treatment enhances collagen-induced MMP-2 activation, which is accompanied by the appearance of a surface-labeled M(r) 43,000 form of MT1-MMP. Treatment of cells with a synthetic furin inhibitor, which inhibits processing of the MT1-MMP zymogen, blocks collagen degradation. In contrast, CAL 27 cells do not degrade collagen under either basal or phorbol 12-myristate 13-acetate-stimulated conditions. Although proMT1-MMP (M(r) 63,000/65,000) is detectable in these cells by immunoblot analysis, they express greatly reduced levels of active MT1-MMP on their surfaces relative to SCC-25 and SCC-15 cells. Correspondingly, CAL 27 cells cultured on collagen express neither latent nor active gelatinases. Immunoblots of lysates and conditioned media revealed the constitutive expression of proMMP-1 and proMMP-13 in all three cell lines. We conclude that in the absence of exogenous growth factors or accessory stromal cells, degradation of interstitial collagen by oral squamous cell carcinoma cells requires a threshold level of active MT1-MMP on cell surfaces.