We have previously demonstrated that xyloglucan is synthesized from UDP-glucose and UDP-xylose in the cell-free enzyme system from suspension-cultured soybean cells. To elucidate the regulation mechanism of xyloglucan biosynthesis, we aimed to determine the levels of UDP-glucose and UDP-xylose in soybean cell cytoplasm and characterize sugar nucleotides to identify donors of galactose and fucose for xyloglucan (composed of glucose, xylose, galactose, and fucose). Suspension-cultured soybean cells (600 g, mid-exponential growth) were extracted with two portions of 70% boiling ethanol for 5 min. The extract was applied to a Dowex 1×8 (Cl⁻) column, and nucleotides were eluted with 0.003 N HCl containing 1 M NaCl after washing. Nucleotides were concentrated via charcoal adsorption and eluted with ammonia-ethanol-water (4:50:46), then separated by paper chromatography with n-butanol-ethanol-water (7:4:2). Isolated nucleotides were fractionated on a Dowex AG1×4 (HCOO⁻) column using gradient elution with formic acid system, and sugar compositions were analyzed by gas-liquid chromatography of alditol trifluoroacetates after mild acid hydrolysis. Total nucleotide yield was 444 μmol per 600 g fresh weight cells. Fractions B, C, and E contained ADP-sugars, GDP-sugars, and UDP-sugars, respectively. Fraction E (UDP-sugars) contained glucose, galactose, xylose, arabinose, and rhamnose, with UDP-glucose and UDP-xylose concentrations of approximately 102 μmol and 2.4 μmol per kg fresh weight cells. Fucose was detected in Fraction C (GDP-sugars), indicating UDP-galactose and GDP-fucose may serve as additional glycosyl donors for xyloglucan synthesis.