In multiple sclerosis (MS), the influx of activated T lymphocytes into the brain parenchyma leads to the subsequent damage of oligodendrocytes, the cells that produce central nervous system (CNS) myelin. We report here that interferon β-1b (IFNβ-1b), a drug shown to be efficacious in the treatment of patients with MS, decreases the in vitro migration of activated T lymphocytes through fibronectin (FN), a major component of the basement membrane that surrounds cerebral endothelium. At 1,000 IU/ml, IFNβ-1b reduced the migratory rate to that of unactivated T cells. In contrast, IFNγ at 1,000 IU/ml, which caused a similar decrease (25%) in the proliferation rate of T lymphocytesas IFNβ-1b, did not affect migration. All T-lymphocyte subsets and natural killer (NK) cells were demonstrated by flow cytometry to be equally affected by IFNβ-1b treatment. 125I-Westernblot analyses revealed that IFNβ-1b treatment resulted in a marked reduction of the ability of T cells to cleave FN. The substrate-degrading capability of T lymphocytes was shown to be due predominantly to the activity of a 92-kd matrix metalloproteinase, MMP-9, whose levels were decreased by IFNβ-1b. We suggest that the clinical benefits of IFNβ-1b treatment in MS patients may be in part a result of the ability of this drug to significantly decrease MMP-9 activity, leading to a reduction of T-lymphocyte infiltration into the CNS.