Astrocytes can be induced by interferon-γ (IFN-γ) to express class II major histocompatibility complex (MHC) antigens. This study was undertaken to elucidate the intracellular signaling pathways involved in IFN-γ induction of class II MHC. We examined the effects of Na+/H+ antiporter and protein kinase C (PKC) inhibitors on class II expression and Na+ influx in astrocytes. We found that amiloride and ethyl isopropylamiloride, inhibitors of Na+/H+ exchange, blocked IFN-γ-induced class II gene expression. IFN-γ stimulated Na+ influx, and this increased influx was inhibited by amiloride. Treatment of astrocytes with the PKC inhibitor H7 also blocked the increase in Na+ uptake induced by IFN-γ, indicating that IFN-γ-induced PKC activation is required for subsequent Na+ influx. IFN-γ treatment produced an increase of total PKC activity, which was associated with a rapid translocation of PKC activity from cytosolic to particulate fraction. H7 and another PKC inhibitor, staurosporine, inhibited IFN-γ-induced class II gene expression. However, 4β-phorbol 12β-myristate 13α-acetate, a potent PKC activator, did not affect class II expression. Taken together, our data indicate that both IFN-γ-induced PKC activation and Na+ influx are required for class II MHC expression in astrocytes but that activation of PKC alone is not sufficient for ultimate expression of this gene.