Ag-Ab complexes are cleared from the circulation through a complex system of receptors for the Fc portion of Ig (FcRs). Fc gamma RIII (CD16) is a low affinity FcR for IgG that is composed of two highly homologous Ig-like extracellular domains. Using secondary structure predictions, we located a strongly hydrophilic region in the second Ig-like domain of Fc gamma RIII that is predicted to lie between beta-strands C and C'. Substitutions of seven out of eight amino acids in this region abolished binding to IgG. Substitution of a conformationally adjacent amino acid in a bend just before beta-strand F and an amino acid in the B-C loop also affected ligand binding. However, amino acid substitutions in two different predicted loops in the second Ig-like domain as well as substitutions to three predicted loops in the first Ig-like domain had no effect on function. A chimeric Fc gamma RIII molecule lacking the second Ig-like domain was unable to bind IgG further, suggesting the presence of the binding site in the second domain. Neutralizing mAbs that inhibit Fc gamma RIII interaction with IgG were mapped to the E-F loop in the membrane proximal domain of Fc gamma RIII, providing further evidence of the importance of this region of the molecule in ligand interaction.