The loss of a functional copy of a heterozygous tumor suppressor gene represents an important step during neoplastic transformation. In order to learn more about the genetic events that lead to spontaneous and drug-induced loss of heterozygosity, a diploid Saccharomyces cerevisiae strain was constructed that allows the detection of the loss of a heterozygous gene by means of direct selection. The strain contains a single functional URA3 gene copy inserted at the ADE2 locus located on the right arm of chromosome 15. In addition, the chromosome contains two other phenotypic marker genes, HIS3 which is located distal from URA3, and PHO80 which is closely linked to the centromere. The homologous chromosome lacks all three marker genes. Loss of the heterozygous copy of URA3 can easily be detected by 5-fluoro-orotic acid resistance of the resulting clones. Simple phenotypic tests of the resistant clones further allows one to distinguish whether the loss of the URA3 gene copy occurred by crossing over, chromosomal loss, or point mutation and gene conversion. Loss of heterozygosity was found to be induced in a dose-dependent fashion by UV radiation and by several chemical agents. All the tested mutagens induced loss of heterozygosity predominantly by crossing over.