Some cell cultures synthesize a mitogenic factor (DNASF) following their lytic infection with either HSV-1 or HSV-2 which is shed into the medium. Of six cell lines permissive to HSV infection tested, three produce DNASF (CV1, HF, MCA) and a significantly higher (3H)-TdR incorporation was obtained with indicator cells growing in supernatants of virus-infected cells (SupV+) than in the corresponding supernatants of sham-treated cells (SupV––). Sometimes the values obtained with SupV+ exceed those obtained with cell controls, which demonstrates that DNASF is not simply a nutritional factor. Other cell lines do not produce DNASF (HeLa, Wistar and probably REF) and SupV+ frequently inhibit cell growth. DNASF is genetically nonspecific and all indicator cells tested were stimulated by supernatants which contain the mitogen, including the nonpermissive DC-3F cells. The quantity of DNASF induced is directly proportional to the number of cells in the culture and its production peaks when the lysis of the cells is complete. It is not accumulated inside the cells.