Sulfatide‐reactive CD1d‐restricted natural killer T (NKT) lymphocytes belong to the type II NKT cell subset with diverse TCRs, and have been found to regulate experimental auto‐immune encephalomyelitis, tumor immunity, and experimental hepatitis in murine models. NKT cells can be activated by self‐lipids presented by CD1d, manifested as autoreactivity. The identity of most of these self‐lipids remains unknown. By isolating lipids from a CD1d‐expressing, highly stimulatory antigen presenting cell, we identified isoforms of β‐glucosylceramide (GlcCer), with sphingosine and fatty acid chain lengths of C24:0 and C16:0, that activated a sulfatide‐reactive type II NKT cell hybridoma. A screen of structurally related glycosphingolipids demonstrated β‐galactosylceramide (GalCer) as another ligand, and further, that the lysoforms were the most potent isoform of the glycosphingo‐lipid ligands, followed by isoforms with a long fatty acid chain of C24. Thus, the same type II NKT cell was activated by several ligands, namely sulfatide, GlcCer, and GalCer. However, CD1d‐dependent reactivity to antigen presenting cells lacking all GlcCer‐based glycosphingolipids, or all glycosphingolipids, was maintained. This suggests that other endogenous, nonglycosphingolipid, lipid ligands contribute to steady‐state autoreactivity by type II NKT cells.