The high affinity receptor for IgE (FcεRI) plays an integral role in triggering IgE-mediated hypersensitivity reactions. The IgE-interactive site of human FcεRI has previously been broadly mapped to several large regions in the second extracellular domain (D2) of the α-subunit (FcεRIα). In this study, the IgE binding site of human FcεRIα has been further localized to subregions of D2, and key residues putatively involved in the interaction with IgE have been identified. Chimeric receptors generated between FcεRIα and the functionally distinct but structurally homologous low affinity receptor for IgG (FcγRIIa) have been used to localize two IgE binding regions of FcεRIα to amino acid segments Tyr128-His134 and Lys154- Glu161. Both regions were capable of independently binding IgE upon placement into FcγRIIα. Molecular modeling of the three-dimensional structure of FcεRIα-D2 has suggested that these binding regions correspond to the 'exposed' C'-E and F-G loop regions at the membrane distal portion of the domain. A systematic site-directed mutagenesis strategy, whereby each residue in the Tyr129-His134 and Lys154-Glu161 regions of FcεRIα was replaced with alanine, has identified key residues putatively involved in the interaction with IgE. Substitution of Tyr131, Glu132, Val155, and Asp159 decreased the binding of IgE, whereas substitution of Trp130, Trp156, Tyr160, and Glu161 increased binding. In addition, mutagenesis of residues Trp113, Val115, and Tyr116 in the B-C loop region, which lies adjacent to the C'-E and F-G loops, has suggested Trp113 also contributes to IgE binding, since the substitution of this residue with alanine dramatically reduces binding. This information should prove valuable in the design of strategies to intervene in the FcεRIα-IgE interaction for the possible treatment of IgE-mediated allergic disease.