Water/salt-insoluble wheat proteins have been identified as the most frequent allergenic foodstuffs in patients with food-dependent exercise-induced anaphylaxis (FDEIA) in Japan. However, the specific allergenic proteins in wheat-dependent exercise-induced anaphylaxis have not been well defined. Challenge testing, skin testing and a fluoroenzyme immunoassay were used for diagnosis in two patients suspected by history of having wheat-dependent exercise-induced anaphylaxis. Gel chromatography and IgE immunoblotting followed by N-terminal amino acid sequencing were used to identify the allergenic wheat protein. The challenge test revealed that both patients had FDEIA. The skin tests and the immunoassay results suggested that wheat gluten was the allergen in both patients. Gel chromatography of wheat gluten revealed that the antigens had molecular weights ranging from 40 to 250 kDa. IgE immunoblotting and subsequent N-terminal amino acid sequencing revealed that wheat-γ-gliadin was the antigen predominantly bound by IgE in the two patients.