The synthesis of herpes simplex virus DNA in isolated nuclei under in vitro conditions was found to be dependent on the addition of ATP and an ATP generating system to the reaction mixture. In vitro DNA synthesis was stimulated and prolonged when p-hydroxymercuribenzoate was added to the isolated nuclei. Under these improved conditions virus DNA molecules which were initiated in vivo were completed in vitro, but most of the DNA molecules synthesized in vitro sedimented in sucrose gradients more slowly than herpes virion DNA. Denaturation of the in vitro labelled DNA molecules produced short single-stranded labelled DNA chains. Thus, under our improved in vitro conditions there was prolonged synthesis of DNA at a high rate, with the formation of both complete and incomplete virus DNA molecules.