Gene targeting with DNA-binding molecules such as triplex-forming oligonucleotides or peptide nucleic acids can be utilized to direct mutagenesis or induce recombination site-specifically. In this chapter, several detailed protocols are described for the design and use of triplex-forming molecules to bind and mediate gene modification at specific chromosomal targets. Target site identification, binding molecule design, as well as various methods to test binding and assess gene modification are described.