Only recently have natural antigens for CD1d-dependent, invariant Valpha14+ natural killer T (iNKT) cells been identified. Similar data for CD1d-independent and CD8+ NKT cell populations are still missing. Here, we show that the MHC class I-restricted CD8+ TCR-transgenic mouse lines OT-I, P14 and H-Y contain a significant proportion of transgenic CD8+ NK1.1+ T cells. In liver, most of NK1.1+ T cells express CD8alphaalpha homodimers. Transgenic NKT cells did not bind invariant Valpha14-to-Jalpha18 TCR rearrangement (Valpha14i)-specific CD1d/alpha-galactosylceramide tetramers and the frequency of iNKT cells was severely reduced. The activated cell surface phenotype and the distribution of transgenic NKT cells in vivo were similar to that reported for iNKT cells. The OT-I and P14 CD8+ NKT cells recognized their cognate antigen in the context of H2-Kb and produced cytokines shortly after TCR stimulation. Importantly, transgenic NKT cells exerted immediate antigen-specific cytotoxicity in vitro and in vivo. Our results demonstrate the presence of transgenic CD8+ NKT cells in MHC class I-restricted TCR-transgenic animals, which are endowed with rapid antigen-specific effector functions. These data imply that experiments studying naive T cell function in TCR-transgenic animals should be interpreted with caution, and that such animals could be utilized for studying CD8+ NKT cell function in an antigen-specific manner.