The anti-CD20 chimeric monoclonal antibody (mAb) rituximab is the most widely used therapeutic antibody for B-cell malignancies. However, approximately 50% of non-Hodgkin's lymphoma (B-NHL) patients respond to treatment with this antibody. Novel humanized antibodies target membrane CD20 with enhanced effector properties should improve treatment for a broader patient population with relapsed and refractory disease. A novel chimerized form of the murine anti-CD20 1K1791 exerts more potent antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities and induces cell death by a non-caspase dependent process. Humanized mAbs derived from 1K1791 were designed using four different humanization techniques and characterized. In contrast to rituximab or 2F2 (human anti-CD20 mAb), several of these exhibited superior ADCC, CDC, inhibition of cell growth and cell death. There was a wide range of functional differences among the humanized forms of 1K1791 despite a modest replacement of amino acid residues in the CDRs. To determine whether the superior activities exhibited by parental murine mAb 1K1791 were due to differences in VH and VL rearrangement, we analyzed its germline and compared it to other anti-CD20 mAbs. A remarkable conservation of VH and Vk (VL kappa) gene usage was observed in the murine anti-CD20 mAbs. 18/23 used the same germline gene J558.42 and 4/23 used closely related genes of the 'J558' group. Thus, 22/23 belonged to VH1 family. One exception was the mAb 1K1791, which was derived from the VH9.12 germline gene. 1K1791 was also unique in its use of a Vk19/28 family gene whereas most other mAbs (21/23) used Vk4/5 family genes. A formal relationship between the particular germline gene recruitment and antibody functionality has not been established, however, the present findings identified humanized mAbs with functional activities that were superior to rituximab and 2F2. These in vitro results support future in vivo animal testing and subsequent clinical trials.