Background: Dust mites are important inducers of allergic disease. Group 2 allergens are recognized as major allergens in several mite species, including Dermatophagoides pteronys-sinus , Lepidoglyphus destructor , and Tyrophagus putrescentiae . No allergens have thus far been characterized on the molecular level from the dust mite Glycyphagus domesticus . Objective: We sought to examine the cross-reactivity among group 2 allergens of G domesticus , L destructor , T putrescentiae , and D pteronyssinus . Methods: A group 2 allergen from G domesticus , Gly d 2, was cloned and expressed as a recombinant protein. Cross-reactivity between Gly d 2 and 3 other group 2 allergens, Lep d 2, Tyr p 2, and Der p 2, was studied by using individual sera and a serum pool RAST-positive to G domesticus , L destructor , T putrescentiae , and D pteronyssinus . Recombinant allergens were used as inhibitors of IgE binding in immunoblotting experiments. Molecular modeling on the basis of the Der p 2 structure was carried out for Gly d 2, Lep d 2, and Tyr p 2. Results: Two cDNAs encoding isoforms of Gly d 2 were isolated, but only the Gly d 2.02 isoform was used in this study. Sixteen of 17 subjects had IgE to Gly d 2. The protein sequence of Gly d 2 revealed 79% identity to Lep d 2 and 46% and 41% identity to Tyr p 2 and Der p 2, respectively. Extensive cross-reactivity was demonstrated among Gly d 2, Lep d 2, and Tyr p 2, but little cross-reactivity was found between these allergens and Der p 2. According to the tertiary structure of Der p 2 and 3-dimensional models of Gly d 2, Lep d 2, and Tyr p 2, differences reside mainly in surface-exposed residues. Conclusion: Gly d 2 showed high sequence homology to Lep d 2. Cross-reactivity was observed between Gly d 2, Lep d 2, and Tyr p 2, but only limited cross-reactivity was demonstrated between these 3 allergens and Der p 2. (J Allergy Clin Immunol 2001;107:511-8.)