Infection of Vero cells with herpes simplex virus type 1 results in the appearance in soluble extracts of a DNA primase activity. The partially purified enzyme, Mr, approximately 100,000, is identical in resistance to alpha-amanitin, pH profile, Mg2+ dependence, salt sensitivity, and KmATP to the catalytic core of Vero cell mitochondrial RNA polymerase. Moreover, the products synthesized are those expected of an RNA polymerase rather than a DNA primase. Inasmuch as the enzyme is not present in soluble extracts of uninfected Vero cells, we presume that the specific appearance of RNA polymerase in extracts of herpesvirus-infected cells results from infection-induced disruption of the mitochondrial membrane, followed by release of the enzyme into the cytosol.