The recently characterized cytosolic transglucosidase DPE2 (EC 220.127.116.11) is essential for the cytosolic metabolism of maltose, an intermediate on the pathway by which starch is converted to sucrose at night. In in vitro assays, the enzyme utilizes glycogen as a glucosyl acceptor but the in vivo acceptor molecules remained unknown. In this communication we present evidence that DPE2 acts on the recently identified cytosolic water-soluble heteroglycans (SHG) as does the cytosolic phosphorylase (EC 18.104.22.168) isoform. By using in vitro two-step 14C labeling assays we demonstrate that the two transferases can utilize the same acceptor sites of the SHG. Cytosolic heteroglycans from a DPE2-deficient Arabidopsis mutant were characterized. Compared with the wild type the glucose content of the heteroglycans was increased. Most of the additional glucosyl residues were found in the outer chains of SHG that are released by an endo-α-arabinanase (EC 22.214.171.124). Additional starch-related mutants were characterized for further analysis of the increased glucosyl content. Based on these data, the cytosolic metabolism of starch-derived carbohydrates is discussed.