Matrix metalloproteinases (MMPs), the key effectors of extracellular matrix remodeling, have been demonstrated to regulate the extension of neurites from neuronal cell bodies. In this report we have addressed the hypothesis that oligodendrocytes (OLs) may utilize a similar mechanism in extending their processes during the initial phase of myelination. Furthermore, given our previous findings linking protein kinase C (PKC) to the OL process outgrowth, we tested the postulate that this signal transduction pathway may regulate MMPs and thus the process outgrowth phenotype. We demonstrate that in response to pharmacologic activators of PKC, cultured human OLs augment their process extension with a concomitant increase in the activity of an MMP, MMP-9, as measured by gelatin zymography. Similarly, the phorbol ester-enhanced process extension and increased MMP-9 activity were both inhibited by calphostin C, a selective PKC inhibitor. Also, MMP inhibitors such as 1,10-phenanthroline and synthetic dipeptides that inactivate the MMP catalytic site negated the 4beta-phorbol-12,13-dibutyrate (PDB)-mediated process extension, further supporting the key role of MMPs in process extension in vitro. Finally, the elevation of MMP-9 protein expression in the mouse corpus callosum, a tissue rich in OL and myelin, coincided with the previously documented temporal increase in myelination that occurs postnatally. Taken together, these data suggest that MMP-9 constitutes an important mediator of OL process outgrowth, and that this protease in turn can be regulated by PKC. The results are relevant not only to the initial steps of myelination during development, but also to the attempted remyelination that has been shown to occur in pathologic conditions such as MS.