This study defines the molecular basis of the FcαRI (CD89):IgA interaction, which is distinct from that of the other leukocyte Fc receptors and their Ig ligands. A comprehensive analysis using both cell-free (biosensor) and cell-based assays was used to define and characterize the IgA binding region of FcαRI. Biosensor analysis of mutant FcαRI proteins showed that residues Y35, Y81, and R82 were essential for IgA binding, and R52 also contributed. The role of the essential residues (Y35 and R82) was confirmed by analysis of mutant receptors expressed on the surface of mammalian cells. These receptors failed to bind IgA, but were detected by the mAb MY43, which blocks IgA binding to FcαRI, indicating that its epitope does not coincide with these IgA binding residues. A homology model of the ectodomains of FcαRI was generated based on the structures of killer Ig-like receptors, which share 30-34% identity with FcαRI. Key structural features of killer Ig-like receptors are appropriately reproduced in the model, including the structural conservation of the interdomain linker and hydrophobic core (residues V17, V97, and W183). In this FcαRI model the residues forming the IgA binding site identified by mutagenesis form a single face near the N-terminus of the receptor, distinct from other leukocyte Fc receptors where ligand binding is in the second domain. This taken together with major differences in kinetics and affinity for IgA:FcαRI interaction that were observed depending on whether FcαRI was immobilized or in solution suggest a mode of interaction unique among the leukocyte receptors.