CD1d1 is a member of a family of lipid Ag-presenting molecules. The cellular ligands associated with CD1d1 were isolated and characterized by biochemical means as an approach to elucidate the mechanism by which CD1 molecules assemble in vivo. Natural ligands of mouse CD1d1 included cellular phosphatidylinositol and phosphatidylinositol-glycans that are synthesized in the endoplasmic reticulum. Further biochemical data revealed that the two CD1d1 mutants, one defective in recycling from-and-to the plasma membrane and the other in efficiently negotiating the secretory pathway, associated with phosphatidylinositol. Thus phosphatidylinositol associated with CD1d1 in the early secretory pathway. Phosphatidylinositol also associated with CD1d1 in Pig-A-deficient cells that are defective in the first glycosylation step of glycosylphosphatidylinositol biosynthesis. Moreover, cellular phosphatidylinositol-glycans are not Vα14Jα15 natural T cell Ags. Therefore, we predict that cellular lipids occlude the hydrophobic Ag-binding groove of CD1 during assembly until they are exchanged for a glycolipid Ag (s) within the recycling compartment for display on the plasma membrane. In this manner, cellular lipids might play a chaperone-like role in the assembly of CD1d1 in vivo, akin to the function of invariant chain in MHC class II assembly.