HLA-DO expression is regulated during B-cell development and activation. To determine whether the level of HLA-DO expression is a helpful marker in the differential diagnosis between florid follicular hyperplasia and follicular lymphoma, we analyzed single-cell suspensions from 29 lymph node specimens by 4-color flow cytometry. Of the 29 specimens, 7 were from patients with florid follicular hyperplasia, in which the clonality of the germinal center B cells (GCBCs) was indeterminate by flow cytometric immunophenotyping. The remaining 22 cases were follicular lymphomas; neoplastic cells in 2 of these cases lacked expression of surface immunoglobulin light chains. The level of HLA-DO expression in the reactive CD10+ GCBCs in florid follicular hyperplasia was markedly down-regulated compared with the CD10- polytypic B cells of the same specimens. In contrast, the level of HLA-DO expression in the CD10+ neoplastic cells in all cases of follicular lymphomas was similar to that of the CD10- polytypic B cells, thus higher than the reactive CD10+ GCBCs. This difference was confirmed by immunohistochemical staining. Our results suggest that HLA-DO is a useful marker to differentiate florid follicular hyperplasia from follicular lymphoma by flow cytometry, particularly when clonality of the CD10+ B cells is in question.