A novel sulfoglycosphingolipid based on the isoglobo-series core structure was isolated from rat kidney and purified by column chromatographies with DEAE-Sephadex and silica beads. The structure was characterized by solvolysis, compositional analysis, proton NMR spectroscopy, Fourier-transform infrared spectroscopy, methylation analysis and liquid secondary ion mass spectrometry (LSIMS). The characteristic fragment ions for a sulfate and a sulfated N-acetylhexosamine were observed in LSIMS spectra. The two-dimensional chemical-shift-correlated spectroscopy (COSY) and nuclear Overhauser enhancement spectroscopy experiments evidenced the presence of a 3-O-sulfated N-acetylgalactosamine and a Gal alpha 1-3Gal structure in the molecule. The major ceramide consisted of 4-hydroxysphinganine linked to a C24 nonhydroxy fatty acid, deduced from both compositional analysis and LSIMS. From the above results, the following structure was established for this glycolipid: HSO3-3GalNAc beta 1-3Gal alpha 1-3Gal beta 1-4Glc beta 1-1Cer, isoglobotetraosylceramide (iGb4Cer) IV3-sulfate. Rat kidney also contained globotetraosylceramide (Gb4Cer) IV3-sulfate which has a carbohydrate core identical to that from human kidney. The yields of iGb4Cer IV3-sulfate and Gb4Cer IV3-sulfate were 0.27 and 0.07 nmol/g wet tissue, respectively.