NK1.1(+)alpha betaTCR(+) (NKT) cells have several important roles including tumor rejection and prevention of autoimmune disease. Although both CD4(+) and CD4(-)CD8(-) double-negative (DN) subsets of NKT cells have been identified, they are usually described as one population. Here, we show that NKT cells are phenotypically, functionally and developmentally heterogeneous, and that three distinct subsets (CD4(+), DN and CD8(+)) are differentially distributed in a tissue-specific fashion. CD8(+) NKT cells are present in all tissues but the thymus, and are highly enriched for CD8alpha(+)beta(-) cells. These subsets differ in their expression of a range of cell surface molecules (Vbeta8, DX5, CD69, CD45RB, Ly6C) and in their ability to produce IL-4 and IFN-gamma, with splenic NKT cell subsets producing lower levels than thymic NKT cells. Developmentally, most CD4(+) and DN NKT cells are thymus dependent, in contrast to CD8(+) NKT cells, and are also present amongst recent thymic emigrants in spleen and liver. TCR Jalpha281-deficient mice show a dramatic deficiency in thymic NKT cells, whereas a significant NKT cell population (enriched for the DN and CD8(+) subsets) is still present in the periphery. Taken together, this study reveals a far greater level of complexity within the NKT cell population than previously recognized.