Although the induction of indoleamine 2, 3-dioxygenase (IDO) by several agents is well established, the mechanisms of its transcriptional regulation and those regulating its function as apoptotic mediator seem to be complex, agent-dependent, and cell type-specific. Besides their pro-apoptotic activity in melanoma cells, both anti-Fas agonist antibody (CH11) and the tumor necrosis factor (TNF)-alpha were found to induce IDO gene expression, the activation of apoptosis signal-regulating kinase (ASK1), and the activation of both c-Jun N-terminal kinase (JNK) and NF-kappaB pathways. In addition, the treatment of melanoma cells with CH11 or TNF-alpha induced the loss of mitochondrial membrane potential (Deltapsim), the accumulation of reactive oxygen species (ROS), the phosphorylation of Fas-associated domain (FADD), the cleavage of caspase-8, and truncation of Bid. Using RNA interference and pharmacological inhibitors, we could confirm the pro-apoptotic activity of IDO and address the mechanisms, which are responsible for its transcriptional regulation and the modulation of its pro-apoptotic activity during death receptor activation in melanoma cells. Thus, our data confirm the pro-apoptotic activity of IDO and provide an insight into the molecular mechanism of TNF-alpha and CH11-induced IDO expression, and the mechanism whereby IDO induces apoptosis of melanoma cells.