The conversion of superhelical (Form I) SV40 DNA to nicked molecular forms was studied by incubating SV40 [3H]DNA for ½ to 2 hr with monolayer cultures of monkey, human, or mouse cells in the presence of DEAE-dextran. The superhelical and the nicked DNA forms were then extracted from the cultures and separated by equilibrium centrifugation in caesium chloride-ethidium bromide (CsCl-EtBr) density gradients. About 66 and 42% of the Form I [3H]DNA was converted to nicked DNA during 2 hr of adsorption to monkey and to human cells, respectively. Form I DNA was adsorbed less by mouse kidney cells than by monkey cells, and only about 25% of the input DNA was converted to nicked forms in 2 hr. The specific infectivities (p.f.u./counts/min.) of the SV40 DNA extracted from the cultures were about the same as those of the input DNA, despite the fact that as much as 69% of the extracted DNA represented nicked molecular forms.