Oral administration of myelin antigens reduces the incidence and severity of EAE in rat and mouse models and decreases the frequency of MBP-reactive cells and the frequency of attacks in some patients with multiple sclerosis. Low-dose oral tolerance has been shown to be mediated by Th2-type regulatory cells that secrete TGFbeta and IL-4/IL-10. Adjuvants and cytokines may modulate oral tolerance. The addition of betaIFN to the experimental therapy regimen, either orally or by intraperitoneal injection, has been shown to enhance the suppressive effects of oral myelin antigens when either are fed the suboptimal dosing regimen to suppress EAE. The current studies were conducted to elucidate the mechanism of the observed in vivo synergy of betaIFN and antigen feeding. Analysis of the in vitro proliferative response and cytokine production by lymphocytes from fed animals in response to specific antigen in culture shows that the synergistic effect may be related to both independent suppression of the immune response by oral betaIFN and enhanced production of TGFbeta and IL-4/IL-10. There was an unexpected increase in the production of gammaIFN by lymphocytes in vitro after three doses of oral betaIFN in vivo. These observations have important implications for the use of cytokines to modulate oral tolerance.