The human neutrophil NB1 glycoprotein (NB1 GP, HNA-2a, CD177) has gained clinical importance for being involved in pulmonary transfusion reactions and immune neutropenias. The NB1 GP shows the unique feature of being expressed only on a neutrophil subpopulation. Recently, we identified splicing defects responsible for an NB1 GP deficiency. In this study, we have investigated the molecular basis of the heterogeneous expression of NB1 GP by separating the 2 neutrophil subpopulations using immunofluorescence followed by single-cell picking or by fluorescence-activated cell sorter. We found a lack of NB1 mRNA in the NB1 GP^- cells that remained constant even after granulocyte colony-stimulating factor (G-CSF) administration. Comparing the cDNA sequences of donors with a large (> 60%) and those with a small (< 40%) NB1 GP–expressing subpopulation, we found 6 polymorphisms. Of the 6, 3 were significantly associated with a small NB1 GP–expressing subpopulation, indicating a genetic basis for NB1 GP nonexpression.