A five-sugar sulfated glycosphingolipid containing an isoglobo-series carbohydrate core was isolated from rat kidney and its structure characterized by compositional analysis, FTIR spectroscopy, methylation analysis and 1H NMR spectroscopy of the intact glycolipid and its limited degradation products, and negative liquid secondary ion mass spectrometry (LSIMS). The two dimensional chemical shift correlated spectroscopy and NOE spectroscopy provided information on the sugar sequence and linkage as well as anomeric configurations, so as to establish the presence of a 3-O-sulfated galactose and a Gal alpha 1-3Gal structure within the molecule. Negative LSIMS with collision-induced dissociation defined the sugar sequence and ceramide composition, allowing to confirm the presence, and indicating the position, of the sulfate group. The glycosphingolipid was found to be a mono-sulfated derivative of the isoglobo-series core, with the following structure: HSO3(-)-3Gal beta 1-3GalNAc beta 1-3Gal alpha 1-3Gal beta 1-4Glc beta 1-1Cer (iGb5Cer V3-sulfate).