To generate industrially applicable new host cell lines for antibody production with optimizing antibody‐dependent cellular cytotoxicity (ADCC) we disrupted both FUT8 alleles in a Chinese hamster ovary (CHO)/DG44 cell line by sequential homologous recombination. FUT8 encodes an α‐1,6‐fucosyltransferase that catalyzes the transfer of fucose from GDP‐fucose to N‐acetylglucosamine (GlcNAc) in an α‐1,6 linkage. FUT8^−/− cell lines have morphology and growth kinetics similar to those of the parent, and produce completely defucosylated recombinant antibodies. FUT8^−/−‐produced chimeric anti‐CD20 IgG1 shows the same level of antigen‐binding activity and complement‐dependent cytotoxicity (CDC) as the FUT8^+/+‐produced, comparable antibody, Rituxan. In contrast, FUT8^−/−‐produced anti‐CD20 IgG1 strongly binds to human Fcγ‐receptor IIIa (FcγRIIIa) and dramatically enhances ADCC to approximately 100‐fold that of Rituxan. Our results demonstrate that FUT8^−/− cells are ideal host cell lines to stably produce completely defucosylated high‐ADCC antibodies with fixed quality and efficacy for therapeutic use. © 2004 Wiley Periodicals, Inc.