Recent studies have revealed different acrylamide formation mechanisms, e. g. from carnosine (N-beta-alanyl-L-histidine) and aminopropionamide as additional precursors. The occurrence of acrylamide in food matrices devoid of common precursors such as meat supports an additional formation pathway. Gluten was recovered from wheat flour by water extraction. Starch, reducing sugars and amino acids were removed using alpha-amylase and NaCl solution and were completely absent in the purified gluten fraction. The gluten was dry heated at temperatures ranging from 160 to 240 degrees C for 8 to 12 min and analyzed for acrylamide and cinnamic amide using liquid chromatography-tandem mass spectrometry. Acrylamide could be detected up to 3997 microg/kg gluten dry weight. Cinnamic amide was detected and unambiguously identified in the gluten samples, thus confirming the proposed formation of acrylamide from proteins. After gluten addition to bread roll dough, protein pyrolysis to form acrylamide in the complex food matrix was assessed. Contents of asparagine and reducing sugars were diminished due to the addition of the gluten. In contrast to the expectation with respect to the well-established common formation mechanism of acrylamide, it increased from 53.4 to 63.9 microg/kg (+20%), which was in good correlation with the higher proportion of gluten. As demonstrated by the t-test, the increase in acrylamide was significant when comparing 0 and 15% gluten addition. Additionally, cinnamic amide could be found in crusts of bread rolls. Thus, evidence for pyrolytic formation of acrylamide from wheat gluten was provided.