Glutathione status and its relationship to molecular weight distribution (MWD) of wheat polymeric protein (PP) during the grain development have been examined. Changes in reduced glutathione (GSH), glutathione disulfide (GSSG), and protein-glutathione mixed disulfide (PSSG) levels were followed through reversed phase high performance liquid chromatography (RP-HPLC) analysis. By applying this procedure to two varieties of wheat, differing in high molecular weight glutenin subunit (HMW-GS) composition (2*, 7 + 8, 5 + 10 and 0, 6 + 8, 2 + 12, respectively, for Soissons and Thésée), we showed that during the grain desiccation (after 33 d after anthesis) which coincide with the formation of SDS-unextractable polymeric protein (UPP), glutathione reductase activity declines, the ratio of GSSG to GSH increases and PSSG occurs. Moreover, approximately 85% of PSSG in mature wheat grains are represented by polymeric protein conjugated to glutathione (PP-S-S-G). As asymmetrical flow field flow fractionation-multi-angle laser light scattering (AFFFF-MALLS) analysis showed, the PP-S-S-G formation during the grain desiccation can induce an increase in the SDS solubility of the polymeric protein by reducing both their molecular weight distribution and the branching of these aggregates. We speculate that glutathione may play a crucial role in controlling the degree of polymerisation of the polymeric protein, which has been shown to be extremely important in determining baking performance.