Since the biologic behavior and molecular genetic changes observed in testicular germ cell cancer differ from those seen in more common epithelial tumors, it is likely that hitherto uncharacterized genes play a role in the development of germ cell tumors. Our previous work on testicular germ cell cancer suggested that chromosome 5q might contain one or more novel tumor suppressor genes that play a role in this malignancy. In this study, we performed a high resolution loss of heterozygosity (LOH) study of testicular cancer using 37 informative markers on chromosome 5. We detected allelic losses in 20/48 (42%) specimens and identified three common sites of loss on chromosome 5q14, 5q21 and 5q34-qter, defined respectively by minimal regions of deletion of < or = 1 cM, 10 cM and approximately 20 (cM). Using an overlapping series of YACs and radiation hybrid mapping, we have constructed a physical map of the 5q14 deletion that should aid in the isolation and characterization of the putative tumor suppressor gene located therein.