Humans and mice contain significant populations of T cells that are reactive for autologous CD1 molecules. Using a panel of five mouse CD1 (mCD1)-autoreactive T cell hybridomas, we show here that this autoreactivity does not correlate with the level of CD1 expression. In some cases, these autoreactive T cells can distinguish between different cell types that express the same CD1 molecule, suggesting that some factor in addition to CD1 expression is critical for autoreactive T cell stimulation. To determine whether a CD1-bound ligand may be required, we expressed mutant mCD1 molecules that are defective for the putative endosomal localization sequence in the cytoplasmic domain. We demonstrate that mCD1, like its human CD1 homologues, is found in endosomes, and that it colocalizes extensively with the DM molecule. We further demonstrate, by site-directed mutagenesis, that the tyrosine in the cytoplasmic sequence is required for this endosomal localization. AT cell hybrid expressing Vβ8 and Vα14, the major TCR expressed by NK1+ T cells, exhibited greatly diminished reactivity to mutant CD1 molecules that do not traffic through endosomes, although the reactivity of other T cell hybrids to this mutant was not greatly affected. Therefore, we propose that at least some of the autoreactive T cells require endosomally derived CD1-bound ligands, and that they are capable of distinguishing between a diverse set of such self-ligands, which might be either autologous lipoglycans or peptides.