We report that a cloned 1620-base-pair (bp) DNA fragment mapping in the BamHI O fragment of herpes simplex virus 1 DNA is amplified after transfection into uninfected cells. The DNA fragment maps entirely within a portion of the open reading frame encoding the large subunit of the viral ribonucleotide reductase and does not contain any of the known lytic origins of viral DNA synthesis. Amplification of this sequence in transfected cells results in accumulation of full-sized Dpn I-resistant plasmids containing the sequence in Hirt extracts of low molecular weight DNA. Subfragments of the 1620-bp fragment were not amplified, whereas larger fragments containing the intact 1620-bp fragment were amplified. The amplification of the fragment in MCF7 cells, which express steroid receptors, was stimulated by the addition of estrogen to the medium. Addition of progesterone, dexamethasone, or testosterone was ineffective. The viral genome therefore contains at least one origin of DNA synthesis capable of supporting replication of viral DNA by cellular factors. The existence of such a host origin of DNA replication in the viral genome was predicted by the hypothesis that viral DNA is amplified by cellular enzymes in sensory neurons harboring latent virus; the link between these sequences and amplification of viral DNA during latency remains to be proven.