Matching of human leukocyte antigen (HLA) alleles between donors and recipients plays a major role in hematopoietic stem cell transplantation (HSCT). Null or questionably expressed HLA allelic variants are a major issue in HLA matching, because the aberrant expression of such alleles can have a major impact on the outcome of HSCT and/or its complications such as graft-versus-host disease. The goal of this study was to investigate the potential of a recently developed cytokine-induced secretion assay to differentiate the expression levels of HLA-A32:11Q (questionable) into a null (N) or low (L) expression variant. An amino acid mutation at position 164 of HLA-A32:11Q disrupts the disulfide bridge in the α2 domain. HLA-A32:11Q is not detectable by standard microlymphocytotoxicity assay. To this end, we cloned soluble HLA-A32:11Q and a reference allele (HLA-A32:01) into expression vectors and transfected/transduced HEK293 and K562 cells. Allele-expressing K562 cells were simultaneously transfected/transduced with a β2-microglobulin (B2M)-encoding vector to ensure the intact HLA structure with B2M. After treatment with proinflammatory cytokines, secreted soluble HLA molecules were determined by enzyme-linked immunosorbent assay in the supernatant and intracellular accumulation of the recombinant proteins by flow cytometry. HLA-A32:11Q was nearly undetectable in untreated transfectants. Cytokine treatment increased the secretion of HLA-A32:11Q to detectable levels and resulted in intracellular accumulation of the allele. There was no difference in mRNA transcription between the A32 alleles. On the basis of these results, we recommend reclassification of HLA-A*32:11Q as a low expression (L) variant.