Interferon (IFN)-induced tryptophan degradation, catalyzed by indoleamine 2,3-dioxygenase (IDO), has been shown to mediate antimicrobial activity in epithelial cells. IDO activity has also been augmented in peripheral blood mononuclear cells (PBMC) treated with IFN or interleukin-2 (IL-2). The effector cells in this population have now been further characterized. PBMCs were isolated from normal donors, separated into monocyte and lymphocyte populations by plastic adherence, treated with IFN or IL-2, and cultivated in medium supplemented with [3H]tryptophan. Culture supernatants were collected after a 48-h incubation and fractionated by high-performance liquid chromatography; radioactivity was determined in fractions corresponding to tryptophan and its metabolites. IFN-gamma and IFN-beta induced IDO activity only in monocytes (plastic-adherent, nonspecific esterase-positive PBMCs). The induction of IDO activity by IL-2 required both monocytes and lymphocytes. Interaction was required between these populations for induction of IDO by IL-2, due to production of IFN-gamma by T lymphocytes, with subsequent IFN-gamma-mediated induction of IDO in monocytes. A number of myeloid cell lines as well as monocyte-derived macrophages were also tested for their ability to be induced to degrade tryptophan in response to IFN treatment. Monocyte-derived macrophages were found to retain their capacity to be induced by IFN-gamma and IFN-beta to degrade tryptophan after differentiation, and to possess seven times more IDO activity per cell than IFN-induced monocytes. However, the presence of lipopolysaccharide (LPS) in the culture medium was required for the maximum induction of IDO activity by IFN-beta. Furthermore, higher concentrations of LPS were sufficient to induce IDO activity in macrophages in the absence of exogenous IFN.