A pool of selectedlacticacidbacteria was used to ferment suspensions of ryeflour. Two-dimensional electrophoresis showed that 109 of the 129 ethanol-soluble rye polypeptides were hydrolysed almost totally by lacticacidbacteria. Matrix-assisted laser desorption ionization—time of flight mass spectrometry and reversed-phase high performance liquid chromatography analysis confirmed the hydrolysis of prolamins. After 48 h fermentation, no prolamin polypeptides were recognized by R5-Western analysis. HPLC analysis of glutelin polymers showed a very low bacterial proteolysis but a pH dependent hydrolysis probably due to activation of rye enzymes. Prolamins were extracted from ryeflour and used to produce a peptic–tryptic (PT)-digest for in vitro tests with K 562 (S) sub-clone and Caco-2/TC7 cells of human origin. The PT-digest was also treated with lacticacidbacteria before assay. The Minimal Agglutinating Capacity increased ca. 8-times when K 562 (S) sub-clone cells were exposed to rye PT-digest treated with lacticacidbacteria. Hydrolysis of rye PT-digest by lacticacidbacteria decreased the toxicity of PT-digest itself towards Caco-2/TC7 cells as estimated by cell viability, caspase-3 activity and release of nitric oxide. Rye prolamins and glutelins were extracted from doughs and subjected to PT digestion. Compared to PT-digests from chemically acidified dough, coeliac jejunal biopsies exposed to the PT-digest from the dough fermented by lacticacidbacteria did not show an increase of the infiltration of CD3+ intraepithelial lymphocytes. The same was found for epithelial cell Fas expression. Long-time fermentation of dough by selectedlacticacidbacteria could be considered as a potential tool to decrease the risk of rye contamination of gluten-free products for coeliac patients.