Gliadin quantification is of essential importance in foods for coeliac patients. Quantification of gliadins is affected by the thermal treatments usually employed in food processing. In order to study the influence of heating on immunochemical detection of gliadin, samples of wheat flour and dough were heated under different time‐temperature conditions (20 min at 100, 130 or 160°C). Ethanolic extracts (70%, ethanol/water, v/v) of treated samples were analyzed by electrophoresis in polyacrylamide gel at pH 3.1 (A‐PAGE) and the protein content quantified. Gliadin was measured by a competitive ELISA. Flour samples suffered no changes in the amount of protein extracted with 70% ethanol nor in the electrophoretic pattern of the extracts after treatments. A rise of 40% in immunochemical reactivity was observed when flour samples heated at 100 and 130°C were analyzed. Higher temperatures produced a drop in reactivity (20% below the original levels). When dough samples were analyzed, an important decrease in extracted protein was observed after the 130 and 160°C treatments. The electrophoretic pattern remained unchanged. A decrease of almost 90% in immunochemical reactivity was also observed after the 130 and 160°C treatments. Our results show that changes in the solubility and immunochemical reactivity of prolamins can lead to an over‐ or under‐estimation of the gliadin content depending on the thermal history and the nature of the sample.