Mice genetically transmitting the exogenous Moloney leukemia virus (Balb/Mo) have been previously derived. These animals carried one copy of Moloney virus DNA (M-MuLV) in their germ line and transmitted the virus as a single Mendelian gene to the next generation. Homozygous BALB/Mo mice were used to genetically map the M-MuLV locus. Embryo fibroblasts were fused to established Chinese hamster cells and somatic cell hybrids were selected. Segregation of mouse chromosomal markers in the hybrids was correlated to the loss of M-MuLV-specific sequences as detected by molecular hybridization. Of 15 isozymes located on different mouse chromosomes only triosephosphate isomerase segregated syntenic with the M-MuLV gene, suggesting that the virus was integrated on chromosome No. 6. This was confirmed by sexual genetic experiments analyzing segregation of Moloney viremia and two markers on chromosome 6 and 15, respectively. The results show that M-MuLV expression is linked to wa-1 on chromosome 6 at a distance of about 30 map units. These data define a new genetic locus, Mov-1, representing the structural gene of M-MuLV in BALB/Mo mice.