We have developed a novel version of the Achilles' Cleavage (AC) reaction in which virtually any restriction site on DNA of any size can be converted to a unique cleavage site. We first polymerized RecA protein on a synthetic oligodeoxyribonucleotide (oligo) in the presence of a nonhydrolyzable ATP analogue to generate oligo:RecA nucleoprotein filaments. These filament were then incubated with plasmid or intact chromosomal DNA from Saccharomyces cerevisiae to form stable complexes in the yeast LEU2 gene at the target sequence identical (or complementary) to that of the oligo. When Hhall (Hinfl) methyltransferase (MH-Hhall) was added, all of the recognition sites for Hhall with the exception of the one protected by the RecA filament were methylated and thus no longer cleaved by the cognate restriction endonuclease (Hinfl). After inactivation of the RecA and the MH-Hhall, Hinfl was used to efficiently cleave the plasmid or chromosome specifically at the targeted restriction site. Since oligos specific for any sequence can be easily synthesized and the other reagents necessary to perform RecA-mediated AC (RecA-AC) reactions on both plasmids and intact chromosomes are readily available, this procedure can be applied immediately to the precise dissection and analysis of genomic DNA from any source and to any other research problem requiring efficient, highly specific cleavage of DNA at predetermined sites.