A serine-containing glycopeptidolipid antigen isolated from Mycobacterium xenopi typified a new class of mycobacterial glycopeptidolipid antigens devoid of the C-mycoside core structure [Rivière, M., & Puzo, G. (1991) J. Biol. Chem. 266, 9057-9063]. The lipopeptide core assigned to C12-Ser-Ser-Phe-alloThr-OCH3 exhibits three potential sites of glycosylation. The carbohydrate parts are composed of 3-O-methyl-6-deoxy-alpha-L-talopyranosyl and 2,3,4-tri-O-methyl-L- rhamnopyranosyl(alpha 1----3)-2-O-lauroyl-L-rhamnopyranosyl(alpha 1----3)-L- rhamnopyranosyl(alpha 1----3)-2,4-di-O-(acetyl, lauroyl)-6-deoxy-alpha-L-glucopyranosyl appendages. In the present work, the carbohydrate attachment sites were successfully determined by ROESY experiments on the native glycopeptidolipid using chloroform as solvent. From the NOE contacts, we unambiguously established that the acylated serine is glycosylated by the 3-O-methyl-6-deoxy-alpha-L-talopyranosyl appendage while the 2,3,4-tri-O-methyl-L-rhamnopyranosyl(alpha 1----3)-2-O- lauroyl-L-rhamnopyranosyl(alpha 1----3)-L-rhamnopyranosyl(alpha 1----3)-2,4-di- O-(acetyl, lauroyl)-6-deoxy-alpha-L-glucopyranosyl appendage is bound to the C-terminal alloThr-OCH3. From these data, the acetyl and lauroyl residues on the C-2 and C-4 of the basal monosaccharide unit were successfully localized. Furthermore, the "L" absolute configuration for the serines and the phenylalanine residues and the "D" configuration for the allothreonine were established. The primary structure of this novel type of mycobacterial antigen, a serine-containing glycopeptidolipid, has now been fully established.