Supernates from myelin basic protein (MBP)-reactive T cell lines have been tested by a battery of assays for cytotoxicity against oligodendrocytes in vitro. All supernates tested demonstrated cytotoxic activity in both a dose- and time-dependent manner that ranged from 29.1% to 55.8% after 48 h incubation. Oligodendrocyte cytotoxicity mediated by MBP-reactive T cell lines was not antigen specific since lines reactive with purified protein derivative (PPD) of tuberculin showed similar cytotoxicity. Supernates cytotoxic to oligodendrocytes were equally effective against syngeneic and non-syngeneic target cells, but had no effect upon astrocytes. Neutralization studies revealed that oligodendrocyte cytotoxicity mediated by MBP-reactive T cell supernates could only be partially attributed to lymphotoxin/tumor necrosis factor activity, and was not associated with perforin, serine proteinase or N-methyl-D-aspartate (NMDA) receptor agonists.