Background: Wheat gluten has unique nutritional and technological characteristics, but is also a major trigger of allergies and intolerances. One of the most severe diseases caused by gluten is coeliac disease. The peptides produced in the digestive tract by the incomplete digestion of gluten proteins trigger the disease. The majority of the epitopes responsible reside in the gliadin fraction of gluten. The location of the multiple gliadin genes in blocks has to date complicated their elimination by classical breeding techniques or by the use of biotechnological tools. As an approach to silence multiple gliadin genes we have produced 38 transgenic lines of bread wheat containing combinations of two endosperm-specific promoters and three different inverted repeat sequences to silence three fractions of gliadins by RNA interference. Results: The effects of the RNA interference constructs on the content of the gluten proteins, total protein and starch, thousand seed weights and SDSS quality tests of flour were analyzed in these transgenic lines in two consecutive years. The characteristics of the inverted repeat sequences were the main factor that determined the efficiency of silencing. The promoter used had less influence on silencing, although a synergy in silencing efficiency was observed when the two promoters were used simultaneously. Genotype and the environment also influenced silencing efficiency. Conclusions: We conclude that to obtain wheat lines with an optimum reduction of toxic gluten epitopes one needs to take into account the factors of inverted repeat sequences design, promoter choice and also the wheat background used.