Cytokine-Ag fusion proteins represent a novel approach for induction of Ag-specific tolerance and may constitute an efficient therapy for autoimmune disease. This study addressed whether a fusion protein containing rat IFN-beta and the encephalitogenic 73-87 determinant of myelin basic protein (i.e., the neuroantigen, or NAg) could prevent or treat experimental autoimmune encephalomyelitis (EAE) in Lewis rats. The optimal structure of the fusion protein was comprised of the rat IFN-beta cytokine as the N-terminal domain with an enterokinase (EK) linker to the NAg domain. Both cytokine and NAg domains had full biological activity. Subcutaneous administration of 1 nmol of IFNbeta-NAg fusion protein in saline on days -21, -14, and -7 before encephalitogenic challenge on day 0 resulted in a substantial attenuation of EAE. In contrast, administration of IFN-beta or NAg alone did not affect susceptibility to EAE. The covalent attachment of IFN-beta and NAg was not necessary, because separate injections of IFN-beta and NAg at adjacent sites were as effective as injection of IFNbeta-NAg for prevention of disease. When treatment was initiated after disease onset, the rank order of inhibitory activity was as follows: the IFNbeta-NAg fusion protein > or = a mixture of IFN-beta plus NAg > IFN-beta > NAg. The novel finding that IFN-beta acts as a tolerogenic adjuvant as well as a tolerogenic fusion partner may have significance for development of tolerogenic vaccines.