The tetrasaccharide 2"-0-(4-0-methyl-β-L-idopyranosyluronic acid) was isolated from enzymatically hydrolysed, unbleached, birch kraft pulp by anion-exchange chromatography in two steps. The primary structure of the tetrasaccharide was determined by 1H and 13C NMR spectroscopy, using homonuclear and heteronuclear two-dimensional techniques. NOE data and 3JH.H coupling constants show that the 4-0-methyl-β-L-idopyranosyluronic acid in the tetrasaccharide is predominantly in the 1C4 chair conformation. The pKa value (3.17) for 4-O-methyliduronic acid attached β-(1 → 2) to xylose was determined from the pH-dependent chemical shift of H-5. The amount of 4-O-methyliduronic acid (0.1–0.5 mol%) in surface xylan of unbleached birch and pine kraft pulps was determined by extensive xylanase treatment and further analysis by NMR spectroscopy and high-performance anion-exchange chromatography.