Atopic allergy is a genetically determined immunodisorder that affects almost 20% of the population worldwide. Immediate symptoms of type I allergy are caused by the release of biologic mediators from effector cells induced by IgE-allergen complexes that cross-link the high-affinity receptor for IgE (FcepsilonRI). Chronic disease manifestations result from allergen-specific T-cell activation, a process that is enhanced when allergens are presented via FcepsilonRI-bound IgE. We report the baculovirus expression, as soluble recombinant proteins, of the minimal units required for human IgE and FcepsilonRI interaction: Cepsilon3 represents the third constant domain of the IgE heavy chain, and alpha2 is the membrane-proximal Ig-like module from FcepsilonRIalpha. Native overlay experiments showed binding of human FcepsilonRIalpha to recombinant Cepsilon3 and of natural or recombinant human IgE to recombinant alpha2. Moreover, recombinant Cepsilon3 inhibited binding of natural IgE antibodies to alpha2, and preincubation of human IgE with alpha2 inhibited anti-IgE-triggered histamine release from human basophils. Isolated Cepsilon3 and alpha2 can now be used for the molecular and structural analysis of the IgE-FcepsilonRI interaction, as well as for diagnostic and therapeutic applications.