Significant proportions of patients with non-Hodgkin lymphoma (NHL), relapsed Hodgkin lymphoma (HL), and chronic lymphocytic leukemia (CLL) are not cured with currently available therapeutic strategies. Recently, a fully human anti-HLA-DR antibody termed 1D09C3 has been generated. It was the aim of the present study to investigate the mechanism of action of 1D09C3 and to further explore in vitro and in vivo its specificity toward neoplastic cells. We investigated a large panel of HLA-DR+ and HLA-DR- cell lines, including CLL (n = 6), NHL (n= 10), and HL (n = 6) cell lines. After treatment with 1D09C3 (2.5 μg/ml, 48 hours), we evaluated viable cell counting by FACS, cell survival by the MTT assay, and apoptosis by annexin-V expression. As compared to controls, exposure of HLA-DR+ cell lines (n = 19) to 1D09C3 reduced the number of viable cells by 4-fold (P ≤0.0001) and cell survival by 2-fold (P ≤0.01), while increasing apoptotic cells by 3-fold (10% vs 33%, P ≤0.0001). No toxicity was observed in HLA-DR- (n = 3) cell lines. Primary normal CD34+ cells, CD14+ cells, NK cells and activated T-lymphocytes were unaffected by 1D09C3 (10 μg/ml), whereas the number of viable B-lymphocytes was reduced by 3-fold. Following exposure of lymphoma cells to 1D09C3, an increase of reactive oxygen species (ROS) was detected. Pre-incubation of JVM-2 cells with the ROS scavenger tiron abolished the effect of 1D09C3 on mitochondrial membrane potential, suggesting that ROS production causes mitochondria involvement in the death process. Western blot analysis showed that constitutive and inducible HSP70 were modulated by 1D09C3, whereas the antibody did not appear to affect Bcl-2 or Bcl-xL. Finally, the activity of 1D09C3 was investigated in vivo in NOD/SCID mice xenografted with JVM-2 (0.25 x 106 cells/mouse, IP) or GRANTA-519 (1 x 106 cells/mouse, IV) cells. Xenografted mice received 1D09C3 (3 x 1 mg/mouse, SC, on days 4, 7, 9) or control vehicle. All placebo-treated mice (n = 15) grafted with JVM-2 died, whereas all 1D09C3-treated mice (n = 15) survived and were disease-free at 120 days (P ≤ 0.0001). All placebo-treated mice (n = 15) inoculated with GRANTA-519 died, whereas treatment with 1D09C3 resulted in a median survival of 90 days with 40% surviving mice which were disease-free at 120 days (P ≤ 0.0001). The efficacy of 1D09C3 was also tested in an advanced disease model. In these experiments, treatment with 1D09C3 (6 x 1 mg/mouse) was started either on day 14 (JVM-2 cells) or day 7 (GRANTA-519 cells). As compared to controls, 1D09C3 injection resulted in a significant prolongation of median survival both for mice xenografted with JVM-2 (29 vs 52 days, P ≤ 0.0001) and GRANTA-519 (35 vs 56 days, P ≤ 0.001). In conclusion, 1D09C3 has no toxic effect on normal primary cells, whereas it has a strong antitumor activity both in vitro and in vivo on HLA-DR+ lymphoma cell lines. Such monoclonal antibody offers the potential for a novel therapeutic approach to lymphoid malignancies.