The transcriptional activation of class II MHC genes requires the class II transactivator (CIITA) protein, a regulator that is essential for both constitutive and IFN-γ-inducible class II MHC expression. The CIITA gene is controlled by multiple independent promoters; two promoters direct constitutive expression, while another, the type IV CIITA promoter, mediates IFN-γ-induced expression. We investigated the molecular regulation of IFN- γ-induced type IV CIITA promoter activity in astrocytes. IFN-γ inducibility of the type IV CIITA promoter is dependent on three cis-acting elements contained within a 154-bp fragment of the promoter; the proximal IFN-γ activation sequence (GAS) element, the E box, and the proximal IFN regulatory factor (IRF) element. Two IFN-γ-activated transcription factors, STAT-1α and IRF-1, bind the proximal GAS and IRF elements, respectively. The E box binds upstream stimulating factor-1 (USF-1), a constitutively expressed transcription factor. Furthermore, STAT-1α binding to the proximal GAS element is dependent on the binding of USF-1 to the adjacent E box. Functionally, the proximal IRF element is essential for IFN-γ induction of type IV CIITA promoter activity, while the proximal GAS and E box elements contribute to the IFN-γ inducibility of this promoter. In astrocytes, TNF- α enhances IFN-γ-induced class II MHC transcription. Our results demonstrate that TNF-α does not enhance IFN-γ-induced transcriptional activation of the type IV CIITA promoter, indicating that the enhancing effect of TNF-α is mediated downstream of CIITA transcription. These results define the molecular basis of IFN-γ activation of the type IV CIITA promoter in astrocytes.