IgG1 antibodies produced in Chinese hamster ovary (CHO) cells are heavily α1,6-fucosylated, a modification that reduces antibody-dependent cellular cytotoxicity (ADCC) and can inhibit therapeutic antibody function in vivo. Addition of fucose is catalyzed by Fut8, a α1,6-fucosyltransferase. FUT8−/− CHO cell lines produce completely nonfucosylated antibodies, but the difficulty of recapitulating the knockout in protein-production cell lines has prevented the widespread adoption of FUT8−/− cells as hosts for antibody production. We have created zinc-finger nucleases (ZFNs) that cleave the FUT8 gene in a region encoding the catalytic core of the enzyme, allowing the functional disruption of FUT8 in any CHO cell line. These reagents produce FUT8−/− CHO cells in 3 weeks at a frequency of 5% in the absence of any selection. Alternately, populations of ZFN-treated cells can be directly selected to give FUT8−/− cell pools in as few as 3 days. To demonstrate the utility of this method in bioprocess, FUT8 was disrupted in a CHO cell line used for stable protein production. ZFN-derived FUT8−/− cell lines were as transfectable as wild-type, had similar or better growth profiles, and produced equivalent amounts of antibody during transient transfection. Antibodies made in these lines completely lacked core fucosylation but had an otherwise normal glycosylation pattern. Cell lines stably expressing a model antibody were made from wild-type and ZFN-generated FUT8−/− cells. Clones from both lines had equivalent titer, specific productivity distributions, and integrated viable cell counts. Antibody titer in the best ZFN-generated FUT8−/− cell lines was fourfold higher than in the best-producing clones of FUT8−/− cells made by standard homologous recombination in a different CHO subtype. These data demonstrate the straightforward, ZFN-mediated transfer of the Fut8− phenotype to a production CHO cell line without adverse phenotypic effects. This process will speed the production of highly active, completely nonfucosylated therapeutic antibodies. Biotechnol. Bioeng. 2010;106: 774–783. © 2010 Wiley Periodicals, Inc.