A GAL1-R/RS-RS cassette DNA was constructed to induce the loss of a specific chromosome in Saccharomyces cerevisiae. The cassette is composed of two specific recombination sites (RS) derived from the pSR1 plasmid of Zygosaccharomyces rouxii, and the site-specific recombinase gene, R, placed downstream of the GAL1 promoter. To delete a certain chromosome, the cassette was inserted into a site on that chromosome in a diploid cell by homologous recombination between a DNA fragment cloned on the cassette and the relevant site on the chromosome. When the transformant was cultivated in galactose medium, elimination of the target chromosome occurred by R-promoted site-specific recombination between two unequal RS sites. Using this method, we demonstrated the loss of chromosomes III, V, VII and XV in heterozygous diploids. Aneuploids appeared at a frequency of 45-85% in the colonies examined after the induction of chromosome loss and were easily distinguished since they gave rise to smaller colonies than did the diploids. We confirmed that the aneuploids often duplicated the residual monosomic chromosome to restore the chromosome balance during mitotic growth. Such diploids formed colonies that were as large as the parent. This method is useful for conversion of heterozygous chromosomes into homozygous (or uniparental) ones in hybrid strains, and is also useful for chromosome loss mapping.