The glycosphingolipid sulfatide (SO₃‐3Galβ1Cer) is a demonstrated ligand for a subset of CD1d‐restricted NKT cells, which could regulate experimental autoimmune encephalomyelitis, a murine model for multiple sclerosis, as well as tumor immunity and experimental hepatitis. Native sulfatide is a mixture of sulfatide isoforms, i.e. sulfatide molecules with different long‐chain bases and fatty acid chain lengths and saturation. Here, we demonstrate that sulfatide‐specific CD1d‐restricted murine NKT hybridomas recognized several different sulfatide isoforms. These included the physiologically relevant isoforms C24:1 and C24:0, major constituents of the myelin sheet of the nervous system, and C16:0, prominent in the pancreatic islet β‐cells. The most potent sulfatide isoform was lysosulfatide (lacking a fatty acid). Shortened fatty acid chain length (C24:1 versus C18:1), or saturation of the long fatty acid (C24:0), resulted in reduced stimulatory capacity, and fatty acid hydroxylation abolished the response. Moreover, sulfatide was not responsible for the natural autoreactivity toward splenocytes by XV19 T hybridoma cells. Our results reveal a promiscuity in the recognition of sulfatide isoforms by a CD1d‐restricted NKT‐cell clone, and suggest that sulfatide, a major component of the myelin sheet and pancreatic β‐cells, is one of several natural ligands for type II CD1d‐restricted NKT cells.