We have recently identified ␣-galactosylceramide (␣-GalCer) as a specific ligand for an invariant V␣14͞ V8.2 T cell receptor exclusively expressed on the majority of V␣14 NKT cells, a novel subset of lymphocytes. Here, we report that ␣-GalCer selectively activates V␣14 NKT cells resulting in prevention of tumor metastasis. The effector mechanisms of the ligand-activated V␣14 NKT cells seem to be mediated by natural killer (NK)-like nonspecific cytotoxicity. Indeed, the cytotoxic index obtained by ␣-GalCeractivated V␣14 NKT cells was reduced by the addition of cold target tumor cells or by treatment with concanamycin A, which inhibits activation and secretion of perforin, but not by mAbs against molecules involved in the NKT cell recognition and conventional cytotoxicity, such as CD1d, V8, NK1.1, Ly49C, Fas, or Fas ligand. These results suggest that the ligand-activated V␣14 NKT cells kill tumor cells directly through a CD1d͞V␣14 T cell receptor-independent, NK-like mechanism.A novel lymphoid lineage, V␣14 natural killer (NK) T cells, distinct from other lymphoid cells including T cells, B cells, and NK cells, is characterized by the early development at day 9.5 of gestation before thymus formation (1) and also by coexpression of the NK receptor and a single, invariant T cell receptor (TCR) encoded by V␣14 and J␣281 gene segments (2-4) in association with a highly skewed set of Vs, mainly V8.2 (5-13). Moreover, the generation of V␣14 NKT cells is exclusively dependent on the expression of the invariant V␣14 TCR as evidenced by the fact that V␣14 NKT cells do not develop in the invariant V␣14 TCR-deficient mice (14) and that the forced expression of the invariant V␣14 TCR leads exclusively to V␣14 NKT cell generation and blocks conventional T cell development (15, 16).Although physiological functions of V␣14 NKT cells remain to be elucidated, the extensive analysis has shown that V␣14 NKT cells are able to mediate allograft bone marrow rejection (17), control autoimmune disease development (18,19), and produce large amounts of both interleukin 4 (IL-4) and interferon ␥ (16,20,21). These findings suggest that V␣14-NKT cells play complicated roles in regulating immune responses more than simply as IL-4 or interferon ␥ providers for Th2 or Th1 cell development, respectively, so far reported (22,23). In addition, we have demonstrated recently that V␣14 NKT cells are a primary target of IL-12 and exert a major effector function in IL-12-mediated tumor rejection (14).A ligand for the invariant V␣14͞V8.2 TCR exclusively expressed on V␣14 NKT cells has been identified recently to be ␣-galactosylceramide (␣-GalCer) (16). In addition, ␣-GalCer is presented by a monomorphic non-majorhistocompatibility gene complex (non-MHC) class Ib molecule, CD1d, expressed on dendritic cells (DC), and the ligand͞CD1 complex selectively stimulates to proliferate V␣14 NKT cells but not other lymphocytes only if costimulatory signals generated by CD40͞CD40 ligand and B7͞ CD28 interactions are provided (16). The results suggest th...