The low‐affinity Fc receptor on human peripheral blood monocytes (FcγRIIA) is polymorphic with respect to its ability to bind murine IgG₁. The two allelic forms of the receptor, high responder (HR) and low responder (LR), yield characteristic patterns after isoelectric focusing and react differently with the anti‐FcγRII monoclonal antibody (mAb), 41H16. We recently cloned cDNA encoding the extracellular domains of FcγRIIA on monocytes from one HR and two LR donors, and found that they differed at only a single base. The cDNA isolated from the HR donor had a G at position 519 and would be expected to encode an aginine at residue 133 in the mature protein, while the cDNA isolated from both LR donors had an A at position 519 and would be expected to encode a histidine at the same residue. To determine whether this single amino acid substitution actually accounts for the functional polymorphism involving FcγRIIA, we transfected COS cells with full‐length HR and LR FcγRIIA cDNA, and examined them for their ability to react with anti‐FcγRIIA mAb and to bind red blood cells (RBC) coated with either murine IgG_2b or murine IgG₁. Whereas COS cells transfected with either the HR cDNA or the LR cDNA reacted with the anti‐FcγRII mAb, IV.3, and bound murine IgG_2b‐coated RBC, only COS cells transfected with the HR cDNA formed rosettes with murine IgG₁‐coated RBC and reacted strongly with mAb 41H16. A total of nine LR donors were identified, and all were homozygous for the A substitution at position 519. We conclude that at an A at position 519 in the cDNA encoding FcγRIIA is the primary molecular basis for the LR form of the receptor, and that the amino acid at residue 133 determines whether FcγRIIA efficiently binds murine IgG₁.