Natural killer (NK) T cells with an invariant V␣14 rearrangement (V␣14i) are the largest population of lipid antigen-specific T lymphocytes identified in animals. They react to the glycolipid ␣-galactosyl ceramide (␣-GalCer) presented by CD1d, and they may have important regulatory functions. It was previously shown that the V␣14i T cell antigen receptor (TCR) has a high affinity for the ␣-GalCer͞CD1d complex, driven by a long half-life (t 1/2 ). Although this result could have reflected the unique attributes of ␣-GalCer, using several related glycolipid compounds, we show here that the threshold for full activation of V␣14i NKT cells by these glycosphingolipids requires a relatively high-affinity TCR interaction with a long t 1/2. Furthermore, our data are consistent with the view that the mechanism of recognition of these compounds presented by CD1d to the V␣14i NKT cell TCR is likely to fit a lock-and-key model. Overall, these findings emphasize the distinct properties of glycosphingolipid antigen recognition by V␣14i NKT cells.