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The hormone, 1,25-dihydroxyvitamin D3 (1,25-[OH]2-D3), inhibits lymphocyte activation in vitro. We studied the ability of the vitamin D metabolite to interfere in vivo with a primary T cell-mediated model of autoimmunity, murine experimental autoimmune encephalomyelitis (EAE). Within 2 wk of antigenic challenge, immunized animals will develop acute paralysis with central nervous tissue inflammation. If mice survive, a rise in antibody titer develops within a month. The administration of 0.1 microgram 1,25-(OH)2-D3 i.p. given every other day for 15 d, starting 3 d before immunization, significantly prevented the development of EAE. The rise in antibody titer to myelin basic protein was also abrogated. Histopathologic lesions of EAE were inhibited by treatment with the sterol. These results suggest a potent immunosuppressive role for 1,25-(OH)2-D3 in vivo in the modulation of a cell-mediated model of autoimmunity.