Human osteosarcomas frequently show loss of alleles on chromosome 17 as well as those on chromosome 13 that harbors the retinoblastoma gene, indicating concerted operation of another tumor-suppressing gene on chromosome 17. To assign the affected gene to a defined region of chromosome 17, we performed mitotic recombination/deletion mapping by the use of 10 polymorphic loci on chromosome 17. Of 37 tumors studied, 28 (75.7%) showed loss of heterozygosity on chromosome 17. The affected regions varied among tumors, ranging in extent from a whole chromosome to a distal segment of the short arm. However, allele loss in one region, notably in 17p13 between D17S1 and D17S30, was common to all 28 tumors, suggesting the presence of a tumor-suppressing gene in this defined region.