Interferon β (IFNβ) is a widespread therapy for multiple sclerosis (MS). We have analyzed some critical features of the T cell activation process in lymph nodes after IFNβ treatment of experimental autoimmune encephalomyelitis (EAE) in SJL mice. Prevention of clinical signs and drastic reduction of perivascular infiltrates in the central nervous system (CNS) were accompanied by alterations in nuclear DNA binding activity levels of NFκB and Stat6 transcription factors in lymph node cells (LNC). A decrease of active NFκB subunits in treated animals correlated with lower levels of the cytoplasmic phosphorylated form of IκBα. Results also showed that nuclear DNA binding activity of Stat6 was increased by IFNβ treatment, as were the cytoplasmic levels of phosphorilated Stat6 (P-Stat6). These high levels of P-Stat6 in IFNβ-treated animals were accompanied by an increase of IL-4 expression levels measured by real time PCR. In vitro experiments with the IL-4 producing clone D10.G4.1 indicates that the IFNβ-mediated IL-4 induction is not an effect exclusive to MBP-reactive cells, and suggest that it could be mediated by mRNA stability enlargement. On the other hand, IFNβ treatment of EAE produced no significant changes in peripheral IFNγ expression and a striking decrease of IL-17. These findings suggest that the inhibition of NFκB activity, the increase of IL-4 expression and its signaling transduction, and the decrease of IL-17 may cooperate to some of the antiinflammatory effects of IFNβ on EAE.