The mechanism of antibody-mediated central nervous system (CNS) demyelination in vivo was studied using rabbit eyes. Injection of anti-galactocerebroside (Gal C) antiserum alone into the normal rabbit vitreous body induced demyelination in the epiretinal myelinated fibers. This activity of the antiserum disappeared after heat treatment at 56 degrees C for 30 min and was restored by supplement of normal fresh serum, suggesting the complement dependency of the activity. Heated anti-Gal C antiserum could induce demyelination, however, when macrophages were introduced by injecting lymphocyte supernatants together with antiserum. Electron microscopic study revealed penetration of macrophage process between the myelin lamella. These findings suggest that the cooperation of anti-Gal C antibody and macrophage can result in the antibody-dependent cell-mediated demyelination in the absence of complement. Because oligodendrocyte generally appeared normal, myelin, not oligodendrocyte is suspected to be the primary target by anti-Gal C antiserum. In contrast, neither anti-MBP nor anti-gangliosides antiserum had the in vivo-demyelinating activity. In CNS demyelination by anti-Gal C antibody, complement-mediated and macrophage-mediated mechanisms may cooperate in varying degrees.