Interferon (IFN)-gamma-induced expression of indoleamine 2,3-dioxygenase (IDO), an enzyme that inhibits some pathogens by limiting tryptophan availability, is transcriptionally enhanced by tumor necrosis factor (TNF)-alpha. The expression of interferon responsive factor (IRF)-1, an IFN-gamma-induced transcriptional activator critical to IDO regulation, is also enhanced synergistically in response to IFN-gamma and TNF-alpha. The IRF-1 regulatory region contains an IFN-gamma-activated sequence (GAS) and a kappaB site, which bind STAT-1 and NF-kappaB, respectively. The TNF-alpha-mediated increase in STAT-1 activation in IFN-gamma-treated cells enhances IRF-1 transcription; however, the contribution of TNF-alpha-mediated increases in nuclear NF-kappaB is uncertain. To identify whether binding of NF-kappaB upstream of the IRF-1 gene is rate-limiting in IRF-1 expression in response to IFN-gamma and TNF-alpha, a proteasome inhibitor was utilized to maintain nuclear translocation of NF-kappaB at constitutive levels; its effect on IRF-1 expression and IDO-specific transcription was evaluated. By limiting NF-kappaB nuclear translocation, IRF-1 expression in IFN-gamma and TNF-alpha treated cells was maintained at a level comparable to that achieved in response to IFN-gamma alone, and the synergistic increase IDO transcription was blocked, suggesting that increases in NF-kappaB translocation are required for synergistic IDO expression in response to IFN-gamma and TNF-alpha.