Eleven purified members of the alpha-amylase/trypsin inhibitor family from wheat and barley that showed very different IgE-binding capacities when previously assayed in vitro, were used in double blind in vivo diagnostic tests to further evaluate their allergenic activity. These tests were carried out in 31 patients who showed allergic sensitization to wheat flour as verified by skin test, RAST and challenge test. The three members of the protein family with highest IgE binding in vitro (the glycosylated subunits of tetrameric alpha-amylase inhibitors CM16* from wheat and CMb* from barley, and the barley monomeric inhibitor BMAI-1) were found to be the strongest allergens as indicated by skin sensitivity in prick tests.