NKT and NK cells are important immune regulatory cells. The only efficient means to selectively stimulate NKT cells in vivo is alpha-galactosylceramide (alphaGalCer). However, alphaGalCer effectively stimulates and then diminishes the number of detectable NKT cells. It also exhibits a potent, indirect ability to activate NK cells. We have now discovered another ceramide compound, beta-galactosylceramide (betaGalCer) (C12), that efficiently diminishes the number of detectable mouse NKT cells in vivo without inducing significant cytokine expression or activation of NK cells. Binding studies using CD1d tetramers loaded with betaGalCer (C12) demonstrated significant but lower intensity binding to NKT cells when compared with alphaGalCer, but both ceramides were equally efficient in reducing the number of NKT cells. However, betaGalCer (C12), in contrast to alphaGalCer, failed to increase NK cell size, number, and cytolytic activity. Also in contrast to alphaGalCer, betaGalCer (C12) is a poor inducer of IFN-gamma, TNF-alpha, GM-CSF, and IL-4 gene expression. These qualitative differences in NKT perturbation/NK activation have important implications for delineating the unique in vivo roles of NKT vs NK cells. Thus, alphaGalCer (which triggers NKT cells and activates NK cells) efficiently increases the resistance to allogeneic bone marrow transplantation while betaGalCer (C12) (which triggers NKT cells but does not activate NK cells) fails to enhance bone marrow graft rejection. Our results show betaGalCer (C12) can effectively discriminate between NKT- and NK-mediated responses in vivo. These results indicate the use of different TCR-binding ceramides can provide a unique approach for understanding the intricate immunoregulatory contributions of these two cell types.