Minicircular SV 40 DNA molecules were obtained by serial undiluted passages of the virus stock. By using high multiplicities of infection, and by cocultivation of infected and uninfected cells, as much as 90% of the SV 40 DNA molecules were deleted. The deletions were confined, as shown by restriction enzyme analysis, preferentially to the late region of the genome. Three size classes of minicircular SV 40 DNA molecules were separated electrophoretically (mini-1, 10–15% deleted; mini-2, 10–20% deleted, and mini-3 20–25% deleted) and used for transfection of permissive CV-1 cells. The cell cultures were examined after various periods of time for the expression of SV 40 tumor and capsid antigens, the replication of viral DNA, the presence of infectious virions and for the rescuability of minicircular DNA after super-infection with wild-type helper SV 40-virus. When helper virus was used to coinfect CV-1 cells together with either mini-1,-2, or-3 DNA, all classes of minicircular DNA were capable of replicating. This complementation effect was abolished, however, in the case of mini-2 and-3-infected CV-1 cells, when the superinfection with helper virus was performed one to two weeks after the initial transfection with minicircular DNA. In the absence of helper virus, only mini-1 DNA could replicate and give rise to infectious virus progeny. Cells which were infected with either mini-2 or-3 DNA displayed no phenotypic alteration for up to one month. Immunofluorescence tests for SV 40 tumor and capsid antigens were negative, and we also could not detect the presence of 3H-labeled superhelical SV 40 DNA. Between 5 and 8 weeks after infection, however, a cytopathic effect developed, and SV 40-gene functions such as virus-specific antigens, replication of SV 40 DNA and appearance of viral progeny were expressed. The size of the newly generated intracellular superhelical SV 40 DNA corresponded predominantly to the size of the minicircular DNA class which had been used for transfection (either mini-2 or mini-3), but larger molecules also appeared (up to the size of wild-type FO I DNA). The restriction enzyme cleavage patterns differed both from the profiles of the wild-type DNA and from mini-2 and mini-3 DNA, respectively; the main difference being that a population of SV 40 genomes had evolved in which sequences from the late region, which were underrepresented in the mini-2 and-3 DNA, had accumulated. This shows that viral DNA molecules containing deletions, which have persisted in the cell for a prolonged period of time in an unexpressed state, can subsequently recombine to form DNA molecules which can give rise to infectious virions.