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Regardless of the input multiplicity used in propagating simian virus 40 in monkey kidney cells, virus populations essentially devoid of defective viruses still contain three types of particles: heavy virus (ρ = 1.35 g/ml), light virus (ρ = 1.325 g/ml) and ‘empty’ capsids (ρ = 1.30 g/ml). Neither light nor heavy virus fractions contain lipids. The protein moiety of the three particle forms is composed of the same polypeptides in similar proportions. DNA extracted from the light virus fraction sediments at a rate identical to that of DNA from heavy virus both in neutral and alkaline sucrose density gradients. The two DNAs hybridize with the same efficiency with the DNA from heavy virus and band together after isopycnic centrifuging in CsCl solution containing ethidium bromide, indicating structural similarity. The sedimentation rates of heavy and light virus particles and their sizes as determined by electron microscopy, are also similar. However, the protein to DNA ratio in the light virus fraction is twice that in the heavy virus fraction. Thus, either heavy virus particles contain two double-stranded, circular DNA molecules of identical molecular size, and light particles contain one, or the light virus fraction contains aggregates of heavy virus and particles devoid of DNA in equal proportion. The specific infectivity of heavy virus is about ten times larger than that of light virus, but the specific infectivity of virus DNA isolated from these virus forms is similar.