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Background: Aspergillus species are prevalent indoor airborne fungi and have been identified to be a causative agent of human allergic disorders. In the present study, we identified, purified and characterized the allergen(s) from Aspergillus flavus, a predominant airborne Aspergillus species in the Taipei area. Methods: The IgE–binding components of A. flavus were identified by SDS–PAGE immunoblotting with sera from asthmatic patients. The N–terminal amino acid sequences of the major allergens were determined by Edman degradation. The allergenic cross–reactivity among allergens from different fungi was analyzed by immunoblot inhibition using sera from asthmatic patients. The detected major allergen was purified from the culture medium. It was further characterized in terms of its N–terminal amino acid sequence, its IgE–binding activity and its enzymatic activity. Results: The results of the immunoblot analysis indicate that a 34–kD component that has high IgE–binding (63%) frequency is a major allergen of A. flavus. The N–terminus of this 34–kD major allergen (GLTTQKSAP) has high sequence identity with that of the 34–kD alkaline serine proteinase major allergen of A. oryzae. Results from immunoblot inhibition studies indicate that IgE cross–reactivity occurs among the 34–kD major allergens of A. flavus, A. fumigatus and Penicillium citrinum. The 34–kD major allergen of A. flavus was purified from the culture medium by ammonium sulfate precipitation and DEAE ion exchange chromatography. The N–terminal amino acid sequence of the purified allergen (Asp fl 13) is identical to that determined previously for the 34–kD major allergen in the crude extract of A. flavus. The IgE immunoblot reactivity to the 34–kD major allergen in the crude extract can be dose–dependently inhibited by the purified Asp fl 13. The degree of IgE binding to the 34–kD major allergen in the crude extract correlates with that of the purified Asp fl 13 in sera of 8 asthmatic patients. The purified Asp fl 13 has proteolytic activity with casein as substrate at pH 8.0. This enzymatic activity can be inhibited by either phenylmethylsulfonyl fluoride or diethylpyrocarbonate. Conclusion: Our results suggest that the 34–kD alkaline serine proteinase is a major allergen of A. flavus. There was IgE cross–reactivity among allergens of A. flavus, A. fumigatus and P. citrinum.