Inactivated herpes simplex virus type 2 (HSV-2) (1, 2) and low concentrations of native, noninactivated viral DNA (3, 4) induce the neoplastic transformation of mammalian cells in culture. Transformation appears to be mediated by fragment(s) of the HSV-2 genome: (i) mechanically sheared HSV-2 DNA (Mr 9 X 106) induces neoplastic transformation at a frequency identical to that observed with native DNA (4), (ii) hamster cells transformed by UV-irradiated HSV-2 retain a fraction (8-32%) of the viral DNA sequences (5), and (iii) virus transforming activity is more resistant than its infectivity to radiation damage (1, 2). However, the oncogenic fragment in the HSV-2 genome has not yet been identified.Provided that oncogenic activity does not depend on distant noncontiguous genes and that suitable restriction endonucleases can yield appropriate fragments of HSV-2 DNA that do not express lytic functions, it seems reasonable to propose that neoplastic transformation should be demonstrable with a restriction endonuclease fragment. The data described in this report support the validity of this proposal.MATERIALS AND METHODS Cells and Virus. Syrian hamster embryo (SHE) cells were grown in ERM medium with 10% fetal bovine serum (3). Vero and HEp-2 cells were grown in medium 199 with 10% fetal bovine serum. HSV-2 strain S-1, a human cervical tumor isolate (6), was plaque-purified and grown in HEp-2 cells at a low (0.1 plaque-forming unit/cell) multiplicity of infection. HSV-2 strain 333 was obtained from R. Duff.DNA Purification. Viral DNA was purified from Vero cells infected with HSV-2 strains S-1 or 333 by buoyant density centrifugation in NaI gradients containing ethidium bromide as described (3, 7). To isolate fragments, we crushed gel slices in buffer (10 mM Tris-HCl/1 mM EDTA/0.1% NaDodSO4, pH 8.0) and incubated them overnight at 45°C. Agarose was removed by centrifugation at 10,000 rpm for 20 min, and the supernatant was extracted with phenol/chloroform/isoamyl alcohol and dialyzed against Hepes-buffered saline, pH 7.05 (9). DNA concentration was determined by UV absorbance at 260 nm. Recovery was approximately 75%.