Successive undiluted passages of SV40 (small plaque type) in VERO cells result in the production of heterogeneous defective particles of lower infectivity than that of infectious virions. It was certified that the infectivity of SV40 ran parallel with the length of its DNA. Thus the defectiveness of virions is due to the decrease in the amount of DNA contained in the virions. Replicating circular molecules of SV40 DNA were isolated from SV40 infected VERO cells. Preparations from dilute and undiluted passaged SV40 virus stocks were shown to give different frequency distributions of contour length of replicating DNA molecules (RF-DNA). The mean length of RF-DNA was 1.26±0.21μ and 1.57±0.81μ for the undiluted and dilute passaged SV40 stocks, respectively. Both types of replicating molecules (θ formand σ form) were noted in both stocks. Small replicating DNA molecules with a contour length less than 1.0μ were noted only in undiluted passaged virus stocks. The presence of shortened replicating forms of viral DNA in undiluted passaged virus stocks would indicate that the DNA of defective SV40 virus can replicate. The DNA isolated from undiluted SV40 virus stocks can transform mouse embryo cells in the presence of 300μg/ml diethylaminoethyl-dextran (DEAE-dextran). T antigen induction was predominant at 5 days after infection, but decreased with increasing time until 30 to 50 days after infection. Numbers of T antigen positive cells increased up to 30% again with morphological changes accompanied. The transformed mouse cells showed criss-cross appearance, pleomorphic nuclei with many nucleolei and irregurality of cell arrangements.