Ninety-five percent of radiation-induced murine myeloid leukemias contain chromosome 2 aberrations. A dominant molecular defect has not yet been identified: both deletions and breakpoint-specific events have been postulated. We have generated a model in which chromosome 2 lesions have been generated in vitro in a clonal tumor cell line. In this study cytogenetic and molecular comparisons are made between two of these in vitro generated lesions and eight derived in vivo: seven by the conventional radiation protocol, and one by infection with Moloney leukemia virus. All 10 lines consistently exhibited hemizygous loss of an 18 cM region between Hoxd-4 and II-1 alpha, with variable breakpoints at both ends. These results are consistent with deletion of a gene in common rather than breakpoint-specific events, for lesions resulting from all three protocols. This will allow a novel approach to the identification of a putative tumor suppressor gene, ie to describe the biological effect of the in vitro generated deletion, and to clone the gene by complementation. In preparation for this approach, we have further narrowed the region to approximately 6.5 cM by microsatellite mapping of 22 radiation-induced F1 tumors. In addition, we have eliminated the possibility that imprinting ablates expression from the remaining undeleted chromosome.