Oligodendrocytes (OLs) extend processes to contact axons as a prerequisite step in myelin formation. As the OL processes migrate toward their axonal targets, they modify adhesion to their substrate, an event that may be regulated by matrix metalloproteinases (MMPs). In the mouse optic nerve, MMP-9/gelatinase B increases during myelin formation. Although tissue inhibitor of metalloproteinase (TIMP)-3 also increases in parallel, the developing optic nerve has focally active MMPs demonstrable by in situ zymography. The distribution of proteolytic activity is similar to that of myelin basic protein, a marker of myelin formation. OLs in culture secrete MMP-9 and express active cell-associated metalloproteinases at the growing tips of their processes. TIMP-1 and a function-perturbing anti-MMP-9 antibody attenuate outgrowth of processes by OLs, indicating a requirement for MMP-9 in process outgrowth. Process reformation is retarded significantly in OLs cultured from MMP-9 null mice, as compared with controls, providing genetic evidence that MMP-9 is necessary for process outgrowth. These data show that MMP-9 facilitates process outgrowth by OLs in vivo and in culture.