Natural killer T (NKT) cells are mainly present in the liver and thymus, and the majority of these T cells express either a CD4(+) or a double-negative (DN) CD4(-)8(-) phenotype. In the present study, we examined whether such NKT cells were present in the intestine. NKT cells were rare in all sites of the small intestine, including an intraepithelial site. However, a considerable number of NKT cells were found at an intraepithelial site in the large intestine. This result was confirmed by both immunofluorescence and immunohistochemistry. In contrast to conventional NKT cells, NKT cells in the large intestine were CD8(+) or DN CD4(-)8(-). In the case of conventional NKT cells, their existence is known to depend on non-classical MHC class I-like antigens (i. e. CD1d) but not on classical MHC class I antigens. However, the NKT cells in the large intestine were independent of the presence of both CD1d and classical MHC class I antigens. These results were obtained using knockout mice lacking the corresponding genes and molecules. NKT cells in the large intestine were mainly alpha betaTCR(+) (> 75 %) but did not use an invariant chain of Valpha14Jalpha281, which is preferentially used by conventional NKT cells. These NKT cells did not bias the TCR-Vbeta usage toward Vbeta8. These findings suggest that the large intestine is a site in which unconventional NKT cells carrying the CD8(+) phenotype (or DN CD4(-)8(-)) are abundant and that these cells are independent of MHC and MHC-like antigens.