A panel of mouse T-cell lymphomas induced by SL3-3 murine leukemia virus (MLV) and three primer binding site mutants thereof (A. H. Lund, J. Schmidt, A. Luz, A. B. Sorensen, M. Duch, and F. S. Pedersen, J. Virol. 73:6117-6122, 1999) were analyzed for the occurrence of recombination between the exogenous input virus and endogenous MLV-like sequences within the 5' leader region. Evidence of recombination within the region studied was found in 14 of 52 tumors analyzed. Sequence analysis of a approximately 330-bp fragment of 44 chimeric proviruses, encompassing the U5, the primer binding site, and the upstream part of the 5' untranslated region, enabled us to map recombination sites, guided by distinct scattered nucleotide differences. In 30 of 44 analyzed sequences, recombination was mapped to a 33-nucleotide similarity window coinciding with the kissing-loop stem-loop motif implicated in dimerization of the diploid genome. Interestingly, the recombination pattern preference found in replication-competent viruses from T-cell tumors is very similar to the pattern previously reported for retroviral vectors in cell culture experiments. The data therefore sustain the hypothesis that the kissing loop, presumably via a role in RNA dimer formation, constitutes a hot spot for reverse transcriptase-mediated recombination in MLV.