Antibody-dependent cellular cytotoxicity (ADCC) and complement fixation both appear to play a role in mediating antitumor effects of monoclonal antibodies (mAbs), including rituximab. We evaluated the relationship between rituximab-induced complement fixation, natural killer (NK)-cell activation, and NK cell-mediated ADCC. Down-modulation of NK-cell CD16 and NK-cell activation induced by rituximab-coated target cells was blocked by human serum but not heat-inactivated serum. This inhibition was also observed in the absence of viable target cells. C1q and C3 in the serum were required for these inhibitory effects, while C5 was not. An antibody that stabilizes C3b on the target cell surface enhanced the inhibition of NK-cell activation induced by rituximab-coated target cells. Binding of NK cells to rituximab-coated plates through CD16 was inhibited by the fixation of complement. C5-depleted serum blocked NK cell-mediated ADCC. These data suggest that C3b deposition induced by rituximab-coated target cells inhibits the interaction between the rituximab Fc and NK-cell CD16, thereby limiting the ability of rituximab-coated target cells to induce NK activation and ADCC. Further studies are needed to define in more detail the impact of complement fixation on ADCC, and whether mAbs that fail to fix complement will be more effective at mediating ADCC.