Gene targeting—the process of gene replacement by homologous recombination—is a very useful but typically inefficient technique (1). A general method for improving the efficiency of gene targeting would be valuable in many circumstances, as would the extension of this genetic tool to a broader range of organisms. Model experiments have demonstrated that a double-strand break (DSB) in the chromosomal target greatly enhances the frequency of localized recombination events (2–6). We have been developing chimeric zinc finger nucleases (ZFNs) as potential stimulators of gene targeting (7, 8). The ZFNs have a DNA recognition domain composed of three Cys2His2 zinc fingers linked to a nonspecific DNA cleavage domain (9). The cleavage domain must dimerize to act as a nuclease (7), and this is most readily achieved for the ZFNs by providing binding sites for two sets of zinc fingers in close proximity and in the appropriate orientations (7, 8). The ability to control the recognition specificity of zinc fingers (10, 11) opens the prospect of directing cleavage to arbitrarily chosen chromosomal sites, without prior manipulation of the target. We designed and constructed a pair of ZFNs (yA, yB) for a site in the second exon of the yellow (y) gene on the X chromosome of Drosophila melanogaster (12) by combining zinc fingers that had been shown in other studies to bind the component triplets of the target sequence (13). Each three-finger DNA binding domain recognizes 9 base pairs (bp). The requirement for dimerization specifies an 18-bp recognition site that is unique in the genome. Previously, we showed that expression of these ZFNs in larvae led to the production of both somatic and germline mutations, due to cleavage and nonhomologous end joining (NHEJ)(12). Here, we tested the ability of ZFN-induced breaks to stimulate gene targeting when a homologous donor DNA is provided. A mutant donor (yM) was constructed, which carries 8 kb of y homology, into which the ZFN recognition site was replaced with two in-frame stop codons and an Xho I site. This mutant was inserted into a P-element vector, flanked by recognition sites for the FLP recombinase and the meganuclease I–Sce I to permit excision and linearization of the donor, and was introduced into the fly genome. Generating a linear extrachromosomal donor DNA in situ by this means has been shown to enhance its effectiveness in recombination (14, 15). The donor was combined by appropriate crosses with transgenes for the yA and yB nucleases and those for FLP and/or I–Sce I, all under the control of the Drosophila HSP70 promoter. Larvae carrying these components were treated by a heat-shock protocol at 35 0 to 4 days after egg laying. The experiment was performed in five versions: ND, no donor: yA yB only; ID, integrated donor: yA yB donor, no FLP or I–Sce I; CD, circular extrachromosomal donor: yA yB FLP donor; LD, linear extrachromosomal donor: yA yB FLP I–Sce I donor; DO, linear donor only: FLP I–Sce I donor, but no ZFNs. Adults emerging from the heat-shock protocol were crossed to reveal germline y mutations (12). In both males and females, the frequencies of mutations rose in the presence of the donor and increased further as it became extrachromosomal and linear (table S1). In the best cases, nearly 20% of males and 14% of females yielded at least one mutant offspring. Chromosomal DNA was recovered from the mutants and analyzed by polymerase chain reaction (PCR), Xho I digestion, and sequence determination (12). Homologous replacements with the donor and the products of NHEJ were identified. The recovery of germline mutants as percent of all offspring …