The role of natural killer T (NKT) cells in the prevention of multiple tumor metastasis was examined. The i.v. inoculation of a highly metastatic subline of B16-BL6 (B16-BL6-HM) melanoma cells resulted in the formation of metastatic nodules in lymph nodes in addition to lung, intrapleural cavity, and ovary. However, treatment of the mice with the NKT cell ligand alpha-galactosylceramide (alpha-GalCer) three times from 1 day after B16-BL6-HM melanoma inoculation caused a significant inhibition of multiple metastasis. Lymph node metastasis of B16-BL6-HM was almost completely blocked by alpha-GalCer treatment. This antimetastatic effect of alpha-GalCer was abolished in NKT cell-deficient mice. These results suggest that alpha-GalCer-activated NKT cells played a critical role in the prevention of lymph node metastasis of melanoma cells.