A single nucleotide polymorphism (SNP) of the FCGR3A gene results in two allotypes of Fcgamma receptor IIIA (FcgammaRIIIA) with valine (V) or phenylalanine (F) at amino acid 158. Since the FcgammaRIIIA-158 V/F polymorphism is associated with the efficacy of monoclonal antibody (mAb) treatment and a risk factor for autoimmune disease, widely applicable methods to assess the SNP are needed. We developed a novel flow cytometric test for this polymorphism using a mAb that recognized only the FcgammaRIIIA-158 V allele (MEM-154) together with a mAb that detected both FcgammaRIIIA-158 alleles (3G8). The expression of both FcgammaRIIIA epitopes on natural killer (NK) cells from 37 healthy donors were measured and compared to the FCGR3A genotype determined by a 5' nuclease assay. FcgammaRIIIA expression levels in individuals with identical FCGR3A genotypes varied considerably, resulting in overlapping immunofluorescences by both 3G8 and MEM-154 between FcgammaRIIIA-158 V/F allotypes. However, the ratio between fluorescences measured using those mAbs in a single individual predicted the FCGR3A genotype with 100% sensitivity and specificity. The novel flow cytometric assay for the FcgammaRIIIA-158 V/F polymorphism that is based on the MEM-154/3G8 fluorescence ratio requires commercially available reagents and a three-color flow cytometer only.