The entire SV40 genome can be recovered from at least some SV40-transformed cell lines which do not sponltaneously produce this virus. Even after repeated cloning in the presence of SV40-antisera, these cell lines can be activated to produce SV40 if they are cocultivated with susceptible monkey kidney cells. Rescue of SV40 is greatly facilitated when mixtures of susceptible and transformed cells are treated with ultraviolet-inactivated Sendai virus (UV-Sendai), a procedure known to produce heterokaryons.1-9 Despite the presence of the entire SV40 genome in transformed cells, Sabin and Koch' failed to detect infectious SV40 DNA. The present study, using an infectious DNA assay of greatly increased sensitivity, also shows that superhelical (closed-circular) SV40 DNA molecules are absent from SV40-transformed cells. Infectious SV40 DNA can, however, be detected after susceptible cells and transformed cells are fused. The time required for emergence of infectious SV40 DNA is significant. T'he data to be presented show that after either fusion of transformed with susceptible cells or productive infection of CV-1 cells, there is about a 16to 19-hour delay before infectious SV40 DNA is made. Also described are attempts to rescue infectious SV40 DNA from transformed cell lines that have so far never yielded virus and from cell lines that are poor virus yielders.9 Materials and Methods. -Cell cultures: CV-1 is an established line of green monkey kidney cells. Transformed mouse kidney lines were obtained by inoculating primary mouse kidney cultures with norrmal SV40 (mKS-A or mKS-BUI00 lines)6 or with SV40 irradiated with UV light to l0oto 10-5 survival (mKS-U lines).' The TSV-5 and the H-50and 2X-10-transformed hamster cells have been described by Tournier et al.5 and by Melnick et al.,10 respectively. All cell lines were grown with R5a medium containing 0.5% lactalbumin hydrolysate and 10% calf serum (Hylands Laboratory, Los Angeles, Calif.). Cell fusion: Approximately 1-1.7 X 107 CV-1 cells were mixed at 4?C for 15 min with 107 transformed cells and 8,000--16,000 hemagglutination units (HAU) of UV-Sendai in a volume of 2 ml. The mixes were shaken for 20 min at 38?C in a water bath, 10 ml of growth medium was added, and the cells were centrifuged and resuspended in 10 ml of growth medium. Two-ml aliquots of cell mixes were seeded in 8-oz prescription bottles, 18 ml of growth medium added, and the cultures were incubated at 37?C for various periods of time. UV inactivation of Sendai virus: Sendai virus was grown in the allantoic cavity of 10to 11-day-old eggs for 48-72 hr at 37?C. After the eggs were chilled at 4?C overnight, the allantoic fluid was har vested arnd clarified by low-speed centrifugation. Sendai virus was pelleted by cenfti,ifugatioti: for 30 n-tin at 30,000 rpim (Spinco model L-2, no. 30 rotor), and the pellets wer e resusp)eided ill 1-)hosphabe-1)lffe le(I saline (1P138) solution'1 at 1/10 the original volume and stored i lthe fro2,en cond(lition. Aliquots were thawed immediately before use and dilulted with 4 vol of PIBS to a titer of 5000-8000 HAU per ml, and 1.6-ml aliquots in 60-mm Petri dishes were inactivated by UV iiradiation for 5 min at a distance of 25 em from the light source (G30T8 Sylvania germicidal lamp).