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 1 H 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 Galα1-3Gal 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:HSO - 3 -3Galβ1-3GalNAcβ1-3Galα1-3Galβ1-4Glcβ1-1Cer (iGb 5 Cer V 3 -sulfate).