SO2-ethanol-water (SEW) lignocellulosic fractionation has the potential to overcome the present techno-economic barriers that hinder the commercial implementation of renewable transportation fuel production. In this study, SEW fractionation of spruce wood chips is examined for its ability to separate the main wood components, hemicelluloses, lignin, and cellulose, and the potential to recover SO2 and ethanol from the spent fractionation liquid. Therefore, overall sulfur and carbohydrate mass balances are established. 95–97 % of the charged SO2 remains in the liquid and can be fully recovered by distillation. During fractionation, hemicelluloses and lignin are effectively dissolved, whereas cellulose is preserved in the solid (fibre) phase. Hemicelluloses are hydrolysed, producing up to 50 % monomeric sugars, whereas dehydration and oxidation of carbohydrates are insignificant. The latter is proven by the closed carbohydrate material balances as well as by the near absence of corresponding by-products (furfural, hydroxymethylfurfural (HMF) and aldonic acids). In addition, acid methanolysis/GC and acid hydrolysis/high performance anion exchange chromatography (HPAEC) methods for the carbohydrate determination are compared.