Visceral leishmaniasis (VL) is a life-threatening disease characterized by uncontrolled parasitization of the spleen, liver, and bone marrow. Interleukin (IL)-10 has been implicated in the suppression of host immunity in human VL based on the elevated levels of IL-10 observed in plasma and lesional tissue, and its role in preventing clearance of Leishmania donovani in murine models of VL. The aim of this study was to identify the cellular source of IL-10 in human VL and determine if CD4(+)CD25(+) (Foxp3(high)) regulatory T (T reg) cells are associated with active disease. We analyzed surface marker and gene expression in peripheral blood mononuclear cells and splenic aspirates from Indian VL patients before and 3-4 wk after treatment with Amphotericin B. The results did not point to an important role for natural CD4(+)CD25(+) (Foxp3(high)) T reg cells in human VL. They did not accumulate in and were not a major source of IL-10 in the spleen, and their removal did not rescue antigen-specific interferon gamma responses. In contrast, splenic T cells depleted of CD25(+) cells expressed the highest levels of IL-10 mRNA and were the predominant lymphocyte population in the VL spleen. The elevated levels of IL-10 in VL plasma significantly enhanced the growth of L. donovani amastigotes in human macrophages. The data implicate IL-10-producing CD25(-)Foxp3(-) T cells in the pathogenesis of human VL.