Interferon (IFN)-γ-induced indoleamine 2,3-dioxygenase (IDO) activity is enhanced synergistically by interleukin (IL-)1, tumor necrosis factor-α (TNF-α) and LPS in IFN-treated macrophages by increasing IDO mRNA concentration. These studies demonstrate that IFN-treated HeLa cells also exhibit dose-dependent enhancement of IDO induction by TNF-α and IL-1, with maximal effects at concentrations of 5ng/ml and 3ng/ml, respectively. Furthermore, with sub-optimal IFN concentrations, cells treated with maximally effective concentrations of TNF-α or IL-1α required 3–5 times less IFN to induce the same level of IDO activity as that observed with IFN alone. To detect changes in transcriptional activation of the IDO gene, HeLa cells were transfected with a plasmid containing the IDO 5′ regulatory region upstream of a green fluorescent protein (GFP) reporter gene. In transfected cells, IFN induced both IDO activity and GFP that was detected by flow cytometry. When cell-sorted, transfected cells were stimulated with IFN in combination with TNF-α or IL-1 but not LPS, increased GFP was detected in comparison to transfected cells treated with IFN alone. Furthermore, increases in GFP expression correlated with IDO enzymatic activity, indicating that combinations of IFN with IL-1 or TNF-α increase the transcriptional activity of the IDO promoter region.