An unusually high incidence of interviral recombination was found in the process of integration of the polyomavirus genome concomitant with neoplastic transformation of nonpermissive cells. Transformants were isolated after mixed infections of Fischer rat cells with two mutants lacking restriction endonuclease sites and were analyzed for the presence of unselected integrated recombinant restriction fragments. A large fraction of the transformants isolated (38% of the 64 transformed cel lines studied) contained recombinant viral genomes that had undergone recombination in a 1.3-, 1.7-, or 3.6-kilobase-pair interval. More than 90% of these recombinant transformants showed evidence of crossovers in multiple intervals. To our knowledge, the recombination frequencies observed in these experiments represent the highest frequencies of homologous recombination reported for a mitotic mammalian system that does not involve transfection. In contrast to the elevated level of recombination in the integrated viral genomes, no evidence of recombination was obtained among the replicated unintegrated pool of viral genomes isolated from the same population of infetted cells from which the recombinant transformants were derived. Either of two hypotheses can provide an explanation for the segregated recombination: either recombination occurs at elevated levels in a small, recombinationprone fraction of the population destined to become transformed, or recombination occurs only among those viral genomes which are engaged in the process of integration and thus interact with a recombinogenic host machinery (for example, the host scaffold). We favor the latter hypothesis.During the events leading to neoplastic transformation of nonpermissive cells by polyomavirus and simian virus (SV40), the viral genome becomes integrated into the host DNA by a mechanism that is facilitated by a viral protein, large T antigen (9, 24). Usually, the integrated viral DNA is present in head-to-tail tandem copies of the viral genome (1, 2, 4), and integration can occur at multiple sites in the genome of the transformed cell (1, 4). Integration occurs by nonhomologous recombination with host DNA and is apparently random with respect to both viral and host sequences (2, 4). At the virus-host junctions, homologies of 2 to 5 base pairs (bp) have been found (5,19,25). Rearrangements and deletions of host DNA are usually associated with the integration events (26,28).Models to explain the tandem topology of integrated viral genomes have invoked a role for either replication (e.g., rolling-circle type) (7, 8) or recombination (11, 15). Basilico and collaborators have shown that the large-T-antigen function (9) and the viral origin of replication (8) are required for tandem formation, consistent with a role for viral DNA replication. A replication step has also been postulated for SV40 by Chia and Rigby (7), based on the existence of unintegrated high-molecular-weight species of viral DNA, which were assumed to represent the unintegrated precursors of the transfor...