To elucidate further the possible role of the tryptophan, rate-limiting enzyme indoleamine 2, 3-dioxygenase (IDO) in leprosy, the distribution of IDOpositive cells and IDO activity in the skin biopsies and sera of these patients representing the entire spectrum of the disease were studied. An increased number of macrophages/dendritic cells (DC–lineage IDO+ cells were found in lepromatous (LL) compared to tuberculoid (BT) and reversal reaction (RR) patients. IDO-positive cells showing CD68 and CD86 surface markers predominated in LL lesions, while higher levels of IDO activity were observed in the sera of LL versus BT patients. Tests revealed an increased IDO message in Mycobacterium leprae-stimulated peripheral blood mononuclear cells (PBMC) by real-time polymerase chain reaction (PCR) and increased IDO expression in M. leprae-stimulated CD14+ cells of both healthy controls (HC) and LL patients, as evaluated via flow cytometry. Increased M. leprae-induced IDO–protein synthesis was also confirmed by Western blot. Based on our in vitro studies, it was confirmed that M. leprae up-regulated IDO expression and activity in HC and LL monocytes. Interferon (IFN)-g synergized with M. leprae in promoting IDO expression and activity in monocytes. IDO expression induced by both IFN-g and M. leprae was abrogated by 1-methyltryptophan (1-MT). Our data suggest that M. leprae chronic infection activates the suppressive molecule IDO which, in turn, contributes to the specific immunosuppression observed in LL leprosy.