Glycolipids with blood group A activity isolated from human and dog small intestine have been characterized by mass spectrometry of intact lipid in methylated and in methylated and reduced (LiAlH4) form. Without degradative studies the glycolipids were conclusively shown to be hexaglycosylceramides with phytosphingosine as the major long-chain base and hydroxypalmitic acid as the major fatty acid. The exact sugar ratio was hexose-hexosamine-deoxyhexose 3:2:1 and the sequence established as hexosamine- [deoxyhexose-] hexose-hexosamine-hexosehexose-ceramide. Evidence is presented that mass spectrometry can differentiate between type 1 and type 2 saccharide chains. © 1975, American Chemical Society. All rights reserved.