Growing evidence indicates antibody-dependent cellular cytotoxicity (ADCC) contributes to the clinical response to monoclonal antibody (mAb) therapy of lymphoma. Recent in vitro analysis suggests C3b can inhibit mAb-induced natural killer (NK) - ell activation and ADCC. Further studies were conducted to assess the effect of C3 depletion on mAb-induced NK activation and therapy of lymphoma. Normal human serum inhibited the ability of rituximab-coated lymphoma cells to activate NK cells as previously reported. Serum did not inhibit NK-cell activation when it was preincubated with cobra venom factor (CVF) to deplete C3. Similar results were found when transudative pleural fluid or nonmalignant ascites was used as surrogates for extravascular fluid, suggesting the inhibitory effect of complement may be present in the extravascular compartment, in which many malignant lymphocytes reside. In vivo, C3 was depleted before mAb treatment in a syngeneic murine model of lymphoma. Survival of lymphoma-bearing mice after treatment with CVF plus mAb and with a human C3 derivative with CVF-like functions (HC3-1496) plus mAb was both superior to that of mAb alone. These studies show that complement depletion enhances NK-cell activation induced by rituximab-coated target cells and improves the efficacy of mAb therapy in a murine lymphoma model.