CD1d-reactive NKT cells are required for the development of antigen-specific T regulatory (Tr) cells responsible for mediating systemic tolerance induced through an immune privileged site such as the eye. The aim of this study was to elucidate the cellular source of CD1d needed for NKT cell activation. Transforming growth factor beta (TGF beta)-2-treated peritoneal exudate cells (PEC) functionally resemble "eye-derived" antigen-presenting cells (APC) and contribute to the generation of Tr cells both in vitro and in vivo. However, when TGF beta-2-treated PEC were pretreated with CD1d-specific antibodies or lacked CD1d expression they failed to induce Tr cells. In addition, we show that the subpopulation of marginal zone (MZ) B cells within the spleen is necessary for induction of Tr cells and that they also must express CD1d. The role of MZ B in tolerance is novel and previously unexplored. Thus, CD1d is necessarily expressed on putative eye-derived APC and splenic MZ B cells for efficient generation of Tr cells in CD1d-reactive NKT cell-dependent tolerance.