Durum wheat contains 10-16% total proteins, where 85% are storage proteins typically divided into monomers (i.e. gliadin) and polymers (i.e. glutenin). Glutenin polymers result from the assembly of these subunits (low and high-molecular weight, LMW and HMW-GS) through inter-chain disulfide bonds. The exceptional rheological properties of wheat gluten depend on the ratio between gliadin/glutenin polymers, but especially on the glutenin size distribution. However, the way glutenin are assembled into polymers is not completely understood. In this study, changes in protein thiol (SH) content, glutenin polymers composition and size distribution were examined in developing durum wheat grains (cv. Orlu, France). Twelve samples of immature grains were monitored until harvest maturity by accumulated temperature/day (°Cd). After SH labeling with a fluorescent alkylant, proteins were sequentially extracted and analyzed by SE-HPLC and SDS-PAGE, applying UV and fluorescence detection. SE-HPLC profiles appeared brightly fluorescent in immature grains (until 473°Cd), indicating a high amount of SH groups. After this, fluorescence declined dramatically and the amount of polymeric protein increased, demonstrating the polymerization comes with oxidation of SH groups. The same results were found by SDS-PAGE. In immature grains, LMW-GS and HMW-GS mainly accumulated as monomers carrying SH groups. The SDS-soluble polymeric fraction included a higher proportion of HMW-GS than LMW-GS, whereas EtOH-soluble polymers consisted mainly of LMW-GS. Subsequently, monomers disappeared to the advantage of polymeric fractions, which still carried some SH groups. To confirm, the ratio Fluorescence/UV210 intensities of SE-HPLC profiles were converted into SH group per subunit involved in polymers, according to the calibration method developed. Our results suggested that glutenin subunits would carry more than two reduced cysteine, indicating the folding of LMW-GS intra-chain disulfide bond might be a late event, posterior to their insertion in a polymeric structure. Assembly of glutenin polymers appears more complex than the linear concatenation model currently accepted.