The expression pattern of mouse CD1d and the tissue distribution of CD1d-restricted Valpha14-Jalpha281 NKT cells suggest that the liver and the marginal zone of the spleen might be preferred sites of activation of this potent innate pathway of early cytokine secretion. Because these tissues are particularly involved with the filtration of blood-borne pathogens, and because NKT cells with an activated / memory phenotype accumulate over the first weeks of life and their CD1 ligands bind microbial glycolipids, it has been hypothesized that expansion of the NKT cell subset may be driven by exposure to the microbial environment. To test this hypothesis, we analyzed the frequency, surface phenotype and functional properties of NKT cells in normal and in germ-free C57BL / 6 mice. Surprisingly, we found that the NKT cell subset develops in the presence or absence of a microbial environment. Although these results do not rule out the possibility that NKT cells exert a protective function against some microbial agents, they demonstrate that non microbial ligands, possibly self-antigens are sufficient for the generation, maturation and peripheral accumulation of NKT cells.