The process of reverse transcription of retroviral genomes begins with the synthesis of a short DNA molecule near the 5' end of the RNA template. This molecule, termed minus-strand strong-stop DNA, is then translocated to the 3' end of the viral RNA by means of a repeated sequence, the R region, present at both ends of the template. The translocation should result in the transfer of genetic information from the 5' R region to the 3' R region. We have generated a series of mutants of Moloney murine leukemia virus with alterations in the R regions by in vitro mutagenesis of a cloned DNA copy of the viral genome. The altered DNAs were introduced into mouse cells by transfection, and the translocation of the mutations during viral replication was assessed. Some mutations were not transferred from the 5' R region to the 3' R region; these results were not in accord with current models for reverse transcription. The results can be explained if DNA molecules shorter than strong-stop DNA, formed by premature termination of synthesis, are sometimes translocated. A number of mutants with large deletions in the R region were tested and were able to replicate with normal strong-stop DNA translocation. Thus, short stretches of homology can be used by the virus to carry out strong-stop translocations.