Interstitial deletion or loss of chromosome 5 is frequent in malignant myeloid disorders, including myelodysplasia (MDS) and acute myeloid leukemia (AML), suggesting the presence of a tumor suppressor gene. Loss of heterozygosity (LOH) analysis was used to define a minimal deletion interval for this gene. Polymorphic markers on 5q31 were identified using a high-resolution physical and radiation hybrid breakpoint map and applied to a patient with AML with a subcytogenetic deletion of 5q. By comparing the DNA from leukemic cells to buccal mucosa cells, LOH was detected with markers D5S476 and D5S1372 with retention of flanking markers D5S500 to D5S594. The D5S500-D5S594 interval, which covers approximately 700 kb, thus represents a minimal localization for the tumor suppressor gene. Further refinement of the physical map enabled the specification of 9 transcription units within the encompassing radiation hybrid bins and 7 in flanking bins. The 9 candidates include genes CDC25, HSPA9, EGR1, CTNNA1, and 5 unknown ESTs. Reverse-transcription polymerase chain reaction confirms that all of them are expressed in normal human bone marrow CD34(+) cells and in AML cell lines and thus represent likely candidates for the MDS-AML tumor suppressor gene at 5q31.