The 15-megabase pair Saccharomyces cerevisiae and the 4.7-megabase pair Escherichia coli genomes were completely cleaved at a single predetermined site by means of the Achilles' heel cleavage (AC) procedure. The symmetric lac operator (lacOs) was introduced into the circular Escherichia coli genome and into one of the 16 yeast chromosomes. Intact chromosomes from the resulting strains were prepared in agarose microbeads and methylated with Hha I (5′-GCGC) methyltransferase (M·HhaI) in the presence of lac repressor (LacI). All Hae II sites (5′-GAGCGC TC) with the exception of the one in lacOs, which was protected by Lacd, were modified and thus no longer recognized by Hae II. After inactivation of M·Hha I and LacI, Hae II was used to completely cleave the chromosomes specifically at the inserted lacOs. These experiments demonstrate the feasibility ofusing the AC approach to efficiently extend the specificity of naturally occurring restriction enzymes and create new tools for the mapping and precise molecular dissection of multimegabase genomes.