A novel method in cleavage of chromosomal DNA at a single site has been developed. Saccharomyces cerevisiae genome was precisely cleaved at a predetermined site using Cre/loxP recombination which mediates the sitespecific recombination between two loxP sites composed of 34bp each. At first, the loxP site was introduced into one of the 16 yeast chromosomes. The chromosomes were isolated from the resulting strain in a intact form and reacted with synthetic oligonucleotides containing loxP sites in the presence of Cre recombinase. By the recombination between two loxP sites on a chromosome and a oligonucleotide, the chromosome containing loxP sites was cleaved specifically. The cleavage was confirmed by Southern hybridization. When biotin-labeled oligonucleotides were used in reaction with plasmid DNA, the simultaneous cutting and labeling of DNA fragment could be accomplished.