Natural killer (NK) T cells with an invariant Valpha14 rearrangement (Valpha14i) are the largest population of lipid antigen-specific T lymphocytes identified in animals. They react to the glycolipid alpha-galactosyl ceramide (alpha-GalCer) presented by CD1d, and they may have important regulatory functions. It was previously shown that the Valpha14i T cell antigen receptor (TCR) has a high affinity for the alpha-GalCer/CD1d complex, driven by a long half-life (t(1/2)). Although this result could have reflected the unique attributes of alpha-GalCer, using several related glycolipid compounds, we show here that the threshold for full activation of Valpha14i 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 Valpha14i NKT cell TCR is likely to fit a lock-and-key model. Overall, these findings emphasize the distinct properties of glycosphingolipid antigen recognition by Valpha14i NKT cells.