Invariant natural killer T (iNKT) cells develop in the thymus upon recognition of CD1d expressed on developing thymocytes. Although CD4 and CD8 coreceptors are not directly involved in the interaction between CD1d and the T cell receptors (TCRs) of iNKT cells, a conspicuous lack of CD8(+) iNKT cells in mice raised the question of whether CD8(+) iNKT cells are excluded due to negative selection during their thymic development, or if there is no lineage commitment for the development of murine CD8(+) iNKT cells. To address this question, we analyzed iNKT cell-specific TCR V alpha 14(+) transgenic mice, where the V alpha 14 transgene forces the generation of iNKT cells. This allows detailed study of the iNKT cell repertoire. We were able to identify CD8(+) iNKT cells which respond to the NKT cell-specific glycolipid ligand alpha-galactosylceramide. Unlike conventional iNKT cells, CD8(+) iNKT cells produce predominantly IFN-gamma but not IL-4 upon antigen stimulation. We also confirmed the presence of CD8(+) iNKT cells in wild type mice. Our results suggest that CD8(+) NKT cells do exist in mice, although their population size is quite small. Their Th1-skewed phenotype might explain why the population size of this subtype needs to be controlled tightly.