From an Aspergillus fumigatus complementary deoxyribonucleic acid (cDNA) library displayed on phage surface, an allergen formally termed rAsp f 3 was cloned. The open-reading frame of the cloned gene for the allergen encodes a protein of 168 amino acids with a predicted molecular mass of 18.5 kD, showing 36% identity and 58% similarity to two peroxisomal membrane proteins of Candida boidinii. Recombinant Asp f 3 was expressed as a [His]6-tagged fusion protein in Escherichia coil at yields of 30 mg/L, and was purified by Ni(2+)-chelate chromatography. In an enzyme-linked immunosorbent assay (ELISA), serum IgE antibody reactivity to rAsp f 3 could be detected in 72% of 89 individuals sensitized to A. fumigatus, demonstrating that the protein represents a major allergen of the mold. IgE specific to rAsp f 3 and the two recombinant Candida proteins was further demonstrated by IgE-immunoblot analysis. IgE binding to rAsp f 3 could be inhibited in the ELISA by adding either of the recombinant Candida peroxisomal proteins to sera containing IgE directed against Asp f 3. Taken together, these observations prove that the Asperigillus allergen and the two Candida proteins share IgE-binding epitopes.