NKT cells respond to a variety of CD1d-restricted glycolipid Ags that are structurally related to the prototypic Ag alpha-galactosylceramide (alpha-GalCer). A modified analog of alpha-GalCer with a carbon-based glycosidic linkage (alpha-C-GalCer) has generated great interest because of its apparent ability to promote prolonged, Th1-biased immune responses. In this study, we report the activation of spleen NKT cells to alpha-C-GalCer, and related C-glycoside ligands, is weaker than that of alpha-GalCer. Furthermore, the Vbeta8.2 and Vbeta7 NKT TCR affinity for CD1d-alpha-C-GalCer, and some related analogs, is approximately 10-fold lower than that for the NKT TCR-CD1d-alpha-GalCer interaction. Nevertheless, the crystal structure of the Vbeta8.2 NKT TCR-CD1d-alpha-C-GalCer complex is similar to that of the corresponding NKT TCR-CD1d-alpha-GalCer complex, although subtle differences at the interface provide a basis for understanding the lower affinity of the NKT TCR-CD1d-alpha-C-GalCer interaction. Our findings support the concept that for CD1d-restricted NKT cells, altered glycolipid ligands can promote markedly different responses while adopting similar TCR-docking topologies.