During demyelinating disease of the central nervous system (CNS), locally elevated cytokine levels may induce upregulation of MHC class II molecules on otherwise low expressing or negative cell types such as microglia and astrocytes, since IFN-gamma has been shown to induce MHC class II expression on these cell types in vitro. While many transcription factors are involved with MHC class II expression, only the class II transactivator (CIITA) is tightly coordinated with IFN-gamma-inducibility. Control of CIITA gene expression is complex, involving four distinct promoters, two of which (promoters III and IV) are IFN-gamma-inducible in certain cell types. Here we demonstrate that IFN-gamma treatment of rat astrocytes induces only CIITA promoter IV activity in contrast to the murine macrophage cell line RAW 264.7 that uses both IFN-gamma-inducible promoters. In contrast to previously published reports, promoter IV activation is completely dependent upon an intact interferon regulatory factor-1 (IRF-1) but not STAT binding site using promoter constructs specifically mutated at these positions. Importantly, while TNF-alpha is able to synergize with IFN-gamma to increase astrocyte MHC class II expression in vitro, we show that treatment of rat astrocytes with TNF-alpha has no effect on CIITA promoter activity. These data demonstrate that TNF-alpha augments MHC class II expression through a mechanism downstream or independent of CIITA induction.