Although complete structures of complex polysaccharides have traditionally been determined by chemical degradative methods, a number of recent developments in instrumentation have greatly facilitated this task. We illustrate the application of several of these methods in a determination of the complete covalent structure of the polysaccharide from Streptococcus sanguis K103, which is composed of an octasaccharide repeating subunit linked by phosphodiester bonds. Carbohydrate analysis by HPAE-PAD and by reverse-phase chromatography of benzoylated derivatives of the hydrolysis products of the polysaccharide gave glucose (3 mol), galactose (1 mol), rhamnose (2 mol), N-acetylglucosamine (1 mol), and galactose 6-phosphate (1 mol). Circular dichroism of the O-benzoylated monosaccharides showed the absolute configurations to be D for all residues except for rhamnose, which is L. The 1H NMR spectrum was completely assigned by two-dimensional homonuclear methods (DQF-COSY, NOESY, HOHAHA). The stereochemistry of pyranosides was assigned from 3JHH coupling constant values determined from these experiments. The 13C spectrum was assigned by 1H-detected heteronuclear multiple-quantum correlation (1H[13C] HMQC) and by the hybrid method of HMQC-COSY. The glycosidic linkage positions of the polymer were determined by 1H-detected multiple-bond correlation (1H[13C] HMBC) and by 2D-NOESY spectra. The position of the phosphodiester linkage was determined by splitting observed in the 13C resonances due to 31P couplings leading to the overall structure given in Chart I.