Hydrolysis and depolymerization of gluten proteins during sourdough fermentation were determined. Neutral and acidified doughs in which microbial growth and metabolism were inhibited were used as controls to take into account the proteolytic activity of cereal enzymes. Doughs were characterized with respect to cell counts, pH, and amino nitrogen concentrations as well as the quantity and size distribution of SDS-soluble proteins. Furthermore, sequential extractions of proteins and analysis by HPLC and SDS-PAGE were carried out. Sourdough fermentation resulted in a solubilization and depolymerization of the gluten macropolymer. This depolymerization of gluten proteins was also observed in acid aseptic doughs, but not in neutral aseptic doughs. Hydrolysis of glutenins and occurrence of hydrolysis products upon sourdough fermentation were observed by electrophoretic analysis. Comparison of sourdoughs with acid control doughs demonstrated that glutenin hydrolysis and gluten depolymerization in sourdough were mainly caused by pH-dependent activation of cereal enzymes.