Activation of invariant natural killer T (iNKT) cells with CD1d-restricted T-cell receptor (TCR) ligands is a powerful means to modulate various immune responses. However, the iNKT-cell response is of limited duration and iNKT cells appear refractory to secondary stimulation. Here we show that the CD94/NKG2A inhibitory receptor plays a critical role in down-regulating iNKT-cell responses. Both TCR and NK-cell receptors expressed by iNKT cells were rapidly down-modulated by priming with a-galactosylceramide (α-GalCer) or its analog OCH [(2S,3S,4R)-1-O-(α-D-galactopyranosyl)-N-tetracosanoyl-2-amino- 1,3,4-nonanetriol)]. TCR and CD28 were re-expressed more rapidly than the inhibitory NK-cell receptors CD94/NKG2A and Ly49, temporally rendering the primed iNKT cells hyperreactive to ligand restimulation. Of interest, α-GalCer was inferior to OCH in priming iNKT cells for subsequent restimulation because α-GalCer-induced interferon γ (IFN-γ) up-regulated Qa-1b expression and Qa-1b in turn inhibited iNKT-cell activity via its interaction with the inhibitory CD94/ NKG2A receptor. Blockade of the CD94/ NKG2-Qa-1b interaction markedly augmented recall and primary responses of iNKT cells. This is the first report to show the critical role for NK-cell receptors in controlling iNKT-cell responses and provides a novel strategy to augment the therapeutic effect of iNKT cells by priming with OCH or blocking of the CD94/ NKG2A inhibitory pathway in clinical applications.