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Polysiphonia nigrescens was sequentially extracted with water at room temperature, 70 and 90 °C. The extracts were analyzed and the major one, isolated at 70 °C, was fractionated by ion-exchange chromatography, eluting with water and solutions of increasing sodium chloride concentration; five main fractions were separated. Structural analysis, carried out by methylation analysis and NMR spectroscopy, showed that four of these were partially cyclized agarans highly substituted on C-6 mainly with sulfate, although methyl ether and single stubs of b-d-xylose were found in minor proportion. A fifth fraction comprising 6-sulfated agarose was also isolated. The use of 2D NMR techniques allowed us to assign the 1H and 13C NMR resonances of the G6S→L6S diad for the first time.