Zinc finger nucleases (ZFNs) are hybrid proteins that have been developed as targetable cleavage reagents for double-stranded DNA, both in vitro and in vivo. This protocol describes the design and construction of new DNA-binding domains comprised of zinc fingers (ZFs) directed at selected DNA sequences. Because the ZFNs must dimerize to cut DNA, they are designed in pairs for any new site. The first step is choosing a DNA segment of interest and searching it for sequences that can be recognized by combinations of existing ZFs. The second step is the construction of coding sequences for the selected ZF sets. Third, these coding sequences are linked to that of the nonspecific cleavage domain from the FokI restriction endonuclease in a cloning vector of choice. Finally, the ZFNs are expressed in Escherichia coli, partially purified, and tested in vitro for cleavage of the target sequences to which they were designed. If all goes smoothly, design, construction and cloning can be completed in about two weeks, with expression and testing completed in one additional week.