Loss of the wild-type allele of a tumor suppressor gene, or loss of heterozygosity (LOH), is one of the most important mechanisms of carcinogenesis. Adenine phosphoribosyltransferase (APRT) has been used as a surrogate marker for tumor suppressor genes. We have previously shown that APRT deficiency in an APRT heterozygous human cell line, MR12-1, was predominantly caused by the loss of the remaining wild-type allele. Here we report the characterization of the chromosomal pathways leading to LOH in four clones derived from this heteroploid cell line. We performed karyotype analysis, chromosome 16-specific painting, and fluorescence in situ hybridization with an APRT-containing cosmid on these clones and their heteroploid parental cells. Our findings suggest that LOH occurs in tetraploid as well as diploid cells, and that diploid cells with LOH may undergo endoreduplication to attain tetraploidy. Our results also suggest that, in addition to LOH being caused by a single event (such as mitotic recombination or deletion), LOH may be caused by a combination of sequential events, such as mitotic recombination or translocation followed by chromosome loss. The instability of the genomes of the parental cells may have provided a greater diversity of options for genetic evolution. Similar karyotypic evolution may occur at late stages of carcinogenesis in vivo.