CD25 (interluekin-2 receptor) expression in diffuse large B-cell lymphoma (DLBCL) cells has been not examined. To characterize CD25(+) DLBCL, 123 patients, who were newly diagnosed with DLBCL, were analyzed by single-color flow cytometry (FCM). CD25-positivity was significantly higher in DLBCL patients (n = 123; mean ± SD, 27.8 ± 30.6%) than in those with reactive lymphadenopathy (n = 16; mean ± SD, 8.6 ± 4.3%) and follicular lymphoma (n = 60; mean ± SD, 12.7 ± 12.4%). By two-color FCM, CD25/CD19 or CD25/CD20 dual positivity in DLBCL patients was shown: mean ± SD, 63.7 ± 25.5% (n = 13) and 55.0 ± 28.1% (n = 14), respectively. Eighty-two percent of the patients with DLBCL received rituximab combined with CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) chemotherapy. A cut-off value of 60% with CD25-positivity clearly divided patients with DLBCL into two groups: CD25-high or CD25-low DLBCL. Although clinical and immunophenotypic features were not significantly different in both groups, the former showed a significantly poorer response and more inferior progression-free survival than the latter. CD25 may be a new prognostic marker and could be a therapeutic target in DLBCL.