Invariant (i) natural killer T (NKT) cells become undetectable after stimulation with alpha-galactosylceramide (alpha-GalCer) or interleukin (IL)-12. Although down-modulation of surface T-cell receptor (TCR)/NKR-P1C (NK1.1) expression has been shown convincingly after stimulation with alpha-GalCer, it is unclear whether this also holds true for IL-12 stimulation. To determine whether failure to detect iNKT cells after IL-12 stimulation is caused by dissociation/internalization of TCR and/or NKR-P1C, or by block of de novo synthesis of these molecules, and to examine the role of IL-12 in the disappearance of iNKT cells after stimulation with alpha-GalCer, surface (s)/cytoplasmic (c) protein expression, as well as messenger RNA (mRNA) expression of TCR/NKR-P1C by iNKT cells after stimulation with alpha-GalCer or IL-12, and the influence of IL-12 neutralization on the down-modulation of sTCR/sNKR-P1C expression by iNKT cells after stimulation with alpha-GalCer were examined. The s/cTCR(+ )s/cNKR-P1C(+) iNKT cells became undetectable after in vivo administration of alpha-GalCer, which was partially prevented by IL-12 neutralization. Whereas s/cNKR-P1C(+) iNKT cells became undetectable after in vivo administration of IL-12, s/cTCR(+) iNKT cells were only marginally affected. mRNA expression of TCR/NKR-P1C remained unaffected by alpha-GalCer or IL-12 treatment, despite the down-modulation of cTCR and/or cNKR-P1C protein expression. By contrast, cTCR(+ )cNKR-P1C(+) sTCR(-) sNKR-P1C(-) iNKT cells and cNKR-P1C(+) sNKR-P1C(-) iNKT cells were detectable after in vitro stimulation with alpha-GalCer and IL-12, respectively. Our results indicate that TCR and NKR-P1C expression by iNKT cells is differentially regulated by signalling through TCR and IL-12R. They also suggest that IL-12 participates, in part, in the disappearance of iNKT cells after stimulation with alpha-GalCer by down-modulating not only sNKR-P1C, but also sTCR.