The mechanism of action underlying the beneficial effect of IFN-beta in multiple sclerosis (MS) is not understood. To date, little information is available on the effects of IFN-beta in experimental autoimmune encephalomyelitis (EAE), the animal correlate of the human disease MS. Therefore, we investigated the effects of recombinant rat IFN-beta (rrIFN-beta) on EAE in Lewis rats with emphasis on a treatment regimen during the paralytic phase of the disease. The results indicated that rrIFN-beta dose-dependently inhibited disease activity with complete prevention at a s.c. dose of 300,000 U/day, provided that treatment was continued for 3 wk. Discontinuation of treatment on day 17 postimmunization resulted in a protracted and relapsing disease course with strongly enhanced clinical severity. Detailed immunohistology of central nervous system (CNS) tissue of protected animals revealed an almost complete absence of CNS lesions and a >90% reduction in the number of infiltrating leukocytes. Accordingly, isolation of mononuclear cells from spinal cord tissue of successfully treated EAE rats revealed a reduction of approximately 95% in the number of cells that produce IFN-gamma in response to the encephalitogenic peptide MBP63-88. Furthermore, rrIFN-beta significantly enhanced serum corticosterone levels, which showed an inverse relationship with disease activity. We show that rrIFN-beta can have both beneficial and detrimental effects on disease activity dependent on the timing and the duration of treatment. Beneficial effects on EAE are associated with inhibition of the extravasation of blood-derived mononuclear cells in the CNS.