When DNA is extracted from mouse cell cultures at various times following infection with polyoma virus, a mixture of both viral and mouse DNA is obtained (Weil, 1963; Dulbecoo, Hartwell & Vogt, 1965; Weil, M&he1 $ Rusohmann, 1965). Viral DNA is only a small fraction of the total DNA (< 15%). Several procedures have been devised for separating polyoma DNA from the cellular DNA. Dulbecco et al.(1965) separated polyoma DNA from mouse DXA by column chromatography using a methylated albumin-kieselguhr complex. A complete separation was not obtained. Sheinin (1966) modified the procedure of Dulbecoo and co-workers by making use of the spontaneous renaturation of polyoma DNA I (20 s) after heat denaturation (Weil, 1963; Weil & Vinograd, 1963). Spontaneously renatured polyoma DNA I was separated from denatured cellular DNA with high resolution by chromatography on methylated albumin-kieselguhr. Since the two minor fractions of polyoma DNA (II and III with sedimentation coefficients of 16 and 14 s) do not renature spontaneously (Weil & Vinograd, 1963), it must be assumed that these components were retained with the denatured cellular DNA under the conditions employed. Weil et al.(1965) obtained partial separation of polyoma DNA from cellular DNA by band sedimentation through CsCl solution. The present communication describes the efllcient separation of viral and cellular DNA by a simple procedure based on the preferential precipitation of undegraded cellular DNA in presence of SDS? and NaCl. Kidney cells from uninfected inbred CR1 mice were grown in modified Eagle’s medium, according to the method of Winocour (1963). The cultures were infected one day after they had reached oonlluence (Petri dishes of 8* 6-cm diameter which contained about lo7 cells). To infect cells, the culture medium was first removed and 0.4 ml. of a suspension containing 4~ 10~ plaque-forming units of polyoma virus (wild type, no. 132, obtained originally from Professor G. Attardi) was added to each culture. After incubation with the viral suspension for one hour, 10 ml. of medium containing 10% horse serum was added. To label the DNA with radioactive isotopes, the medium was replaced at various times after infection by a medium containing [3H] thymidine. After two hours, the radioactive medium was removed, the cells washed twice and incubated with fresh medium for one hour to exhaust the intracellular pool of radioactive thymidine and thymidine phosphates (Weil et al., 1965; Hirt, 1966). Immediately afterwards the medium was removed and the DNA extracted from the cells. 99% of the radioactive material was found in DNA.