Astrocytes are induced by interferon-γ (IFN-γ) to express class II major histocompatibility complex (MHC) antigens. Our previous studies demonstrated that IFN-γ-initiated signaling events important for class II expression include activation of protein kinase C (PKC) and the Na+/H+ antiporter. We have extended these studies and found that protein tyrosine kinase (PTK) activity is also required for class II expression. Treatment of astrocytes with inhibitors specific for PKC and PTK blocked IFN-γ-induced class II gene transcription, mRNA expression, and protein expression. Immunoblotting and immunoprecipitation experiments demonstrated that IFN-γ induced tyrosine phosphorylation of the p91 component of ISGF3, which is blocked by preincubation of cells with PTK inhibitors. Treatment of astrocytes with IFN- γ and either PKC and PTK inhibitors changed the mobility and intensity of a nuclear factor, IFN-γ-enhanced factor X, which binds to the X box of the class II MHC promoter. Taken together, these data provide evidence that activation of both PTK and PKC is required for IFN-γ-induced expression of the class II gene.