Although the p38 mitogen-activated protein kinase (MAPK) has been implicated in signal transduction events, its role in regulating the M_r 92,000 type IV collagenase matrix metalloprotease-9 (MMP-9) and in vitro invasiveness in cancer has not yet been determined. We made the surprising observation that, in a human squamous cell carcinoma cell line (UM-SCC-1), phorbol ester-enhanced MMP-9 secretion and in vitro invasiveness were associated with a strong activation of the p38 MAPK and its downstream target, MAPK-activated protein kinase-2. To determine the role of p38 activation in these events, we investigated the effect of SB 203580, a novel specific p38 inhibitor, on protease expression and in vitro invasion of these cells. We found that inhibition of p38 by SB 203580 resulted in the almost complete reduction of phorbol myristate acetate-induced MMP-9 secretion but not of urokinase-type plasminogen activator secretion. In contrast, the activation of a transiently transfected wild-type MMP-9 promoter by MEKK-1, a specific c-Jun NH₂-terminal kinase activator, was only marginally inhibited by the compound, arguing for the specificity of SB 203580. Moreover, phorbol myristate acetate-enhanced in vitro invasion was completely blocked by SB 203580, whereas p38 inhibition had little effect on growth. These findings suggest that activation of p38 may contribute to a more invasive phenotype in vitro, possibly via the expression of MMP-9, and that targeting of p38 using SB 203580 may provide a novel means of controlling invasion of cancers in which this MAPK is activated.