This study investigates the response of natural killer (NK) T cells to a glycolipid antigen, α-galactosylceramide (α-GalCer), using CD1d tetramers. CD1d tetramers loaded with α-GalCer are shown to be a sensitive and specific reagent for identifying Vα14+ NK T cells. The results indicate that α-GalCer-specific T lymphocytes are more widely distributed than previously thought, with populations of mostly NK1.1+ but tetramer-binding T cells present in lymph nodes and the intestine. Injection of α-GalCer leads to the production of both interferon γ and interleukin 4 by nearly all NK T cells in the liver and the majority of the spleen within 2 hours, but these cells mostly disappear by 5 hours and do not reappear after 1 week. Interestingly, tetramer-positive thymocytes do not rapidly synthesize cytokines or undergo decreases in cell number after lipid antigen stimulation, although they express equivalent TCR levels. The data demonstrate that α-GalCer-specific NK T cells undergo a unique and highly compartmentalized response to antigenic stimulation.This study investigates the response of natural killer (NK) T cells to a glycolipid antigen, α-galactosylceramide (α-GalCer), using CD1d tetramers. CD1d tetramers loaded with α-GalCer are shown to be a sensitive and specific reagent for identifying Vα14+ NK T cells. The results indicate that α-GalCer-specific T lymphocytes are more widely distributed than previously thought, with populations of mostly NK1.1+ but tetramer-binding T cells present in lymph nodes and the intestine. Injection of α-GalCer leads to the production of both interferon γ and interleukin 4 by nearly all NK T cells in the liver and the majority of the spleen within 2 hours, but these cells mostly disappear by 5 hours and do not reappear after 1 week. Interestingly, tetramer-positive thymocytes do not rapidly synthesize cytokines or undergo decreases in cell number after lipid antigen stimulation, although they express equivalent TCR levels. The data demonstrate that α-GalCer-specific NK T cells undergo a unique and highly compartmentalized response to antigenic stimulation.