Previously, we and others identified three subsets of iNKT cells; NKT1, NKT2 and NKT17, which produce distinct cytokines when stimulated. In this study, we defined the anatomic localization and systemic distribution of these subsets, and showed that their localization determines the cytokine response at steady state and upon activation. In the thymus, NKT2 cells that produced IL-4 at steady state were preferentially medullary, where they conditioned medullary thymocytes. In the mesenteric lymph node of BALB/c mice, NKT2 cells were abundant and produced IL-4 in the T cell zone, which conditioned other lymphocytes. Intravenous injection of α-galactosylceramide activated NKT1 cells with vascular access, but not lymph node or thymic NKT cells, resulting in systemic IFN-g and IL-4, while oral α-galactosylceramide activated NKT2 cells in the mesenteric LN, resulting in local IL-4 only.