The structure of the pneumococcal common antigen, C-polysaccharide, from a noncapsulated pneumococcal strain, CSR SCS2, was studied using 1H-NMR, 13C-NMR and 31P-NMR spectroscopy. The dependence of NMR chemical shifts on the variation in pD was also studied. It was established that the C-polysaccharide is composed of a backbone of tetrasaccharide-ribitol repeating units that are linked to each other by a phosphodiester linkage between position 5 of a D-ribitol residue and position 6 of a beta-D-glucopyranosyl residue. The polysaccharide is substituted with one residue of phosphocholine at position 6 of the 4-substituted 2-acetamido-2-deoxy-alpha-D-galactopyranosyl residue. Both galactosamine residues in the polysaccharide are N-acetylated. O)-P-Cho | 6 6)-beta-D-Glcp-(1-->3)-alpha-AATp-(1-->4)-alpha-D-GalpNAc-(1-->3)- bet a-D-GalpNAc-(1-->1)-D-ribitol-5-P-(O--> where AAT is 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose and Cho is choline. This structure differs, concerning phosphocholine substituents and N-acetylation, from those reported previously for pneumococcal C-polysaccharide [Jennings, H.J., Lugowski, C. & Young, N.M. (1980) Biochemistry 19, 4712-4719; Fischer, W., Behr, T., Hartmann, R., Peter-Katalinic, J. & Egge, H. (1993) Eur. J. Biochem. 215, 851-857; Kulakowska, M., Brisson, J.-R., Griffith, D.W., Young, N.M. & Jennings, H.J. (1993) Can. J. Chem. 71, 644-648]. The structures of the C-polysaccharides present in three pneumococcal types were also examined. They contain one (in 18B) or two (in 32F and 32A) phosphocholine residues in the repeating unit. The degree of substitution was not determined. The backbone of all examined C-polysaccharides was identical and in all cases both galactosamine residues appeared to be N-acetylated.