Infection of cell cultures with herpes simples virus type 1 (HSV-1) under standard culture conditions yielded persistently infected cells capable of continued growth in the presence of virus and of forming colonies in agarose. The ability of an infected culture to yield cells able to survive HSV-1 infection was directly related to the presence of S phase cells (cells actively engaged in DNA synthesis) at the time of infection. Only when very high multiplicities of infecting virus (greater than 10) were used did cultures fail to yield persistently infected cells capable of colonial growth in agarose. Cell clones derived from colonies grown in agarose established cell cultures which possessed all the characteristics of HSV-1 persistently infected cultures. When cultures were cloned a second time in agarose, as a rare event, cell cultures which did not immediately liberate infectious virus could be isolated. These cell cultures, however, possessed an increased resistance to superinfection by HSV-1. On continued cultivation they reverted to persistence as exhibited by the sudden onset of virus cytopathic effects and release of infectious virus.