A host protein, which is required for the replication of a plasmid DNA (pLA1), has been purified from extracts of uninfected HeLa nuclei. This plasmid DNA contains the origin of adenovirus DNA replication but lacks the 55,000-dalton terminal proteins. The purified host protein has been designated factor pL. Factor pL is essential for the initiation of DNA replication of EcoRI-digested pLA1 DNA, which proceeds via the formation of a covalent complex between the 80,000-dalton adenovirus coded preterminal protein and 5' dCMP. Factor pL has been purified approximately 120-fold to greater than 75% homogeneity. It is a heat labile and N-ethylmaleimide-sensitive protein with a native Mr = 39,000 (+/- 2,000). Initiation of DNA replication using EcoRI-digested pLA1 DNA as the template requires the 80,000-dalton preterminal protein and the 140,000-dalton adenovirus DNA polymerase, in addition to factor pL, and is stimulated as much as 10-fold by nuclear factor I ( Nagata , K., Guggenheimer , R. A., Enomoto , T., Lichy , J. H., and Hurwitz , J. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6438-6442). Factor pL has no effect on in vitro DNA replication when adenovirus DNA covalently linked to the 55,000-dalton terminal protein is used as the template, however the replication of adenovirus DNA treated with Pronase, becomes totally dependent upon the addition of factor pL.