A polar fucose-containing glycosphingolipid fraction isolated from dog small intestine has been characterized by mass spectrometry of intact methylated, and methylated and reduced (LiAlH4) glycolipid. The native fraction, which was homogenous on thin-layer chromatography, was shown after methylation to be a mixture of two compounds. One was identified as a hexaglycosylceramide with the following composition and sequence: fucose-hexose-[fucose]-hexosamine-hexose-hexose-ceramide, with a terminal saccharide structure similar to blood group Leb determinants. The second compound was a novel heptaglycosylceramide with the sequence: hexosamine-[fucose]-hexose-[fucose]-hexosamine-hexose-hexose-ceramide. This glycolipid was also detected in human small intestine and pancreas. The dog intestinal fraction had phytosphingosine as its major base and contained almost exclusively 2-hydroxy fatty acids (16: 0-24: 0). The fraction of human pancreas differed in having sphingosine as its major base and normal fatty acids (16: 0-24: 0) as major acids. © 1972.