A soluble replication system that synthesizes full-sized adenovirus DNA molecules has been developed. The extraction of infected HeLa nuclei solubilizes approximately 25% of the viral replicating DNA and leaves the HeLa chromatin associated with the insoluble nuclear material. Both the extracted viral DNA and the DNA product synthesized in vitro are intact and identical to the adenovirus DNA or its replicating intermediates produced in whole infected cells. The direction of elongation and the termini of replication of progeny DNA in vitro are essentially identical to those observed in vivo. Nuclear extract replication is dependent on Mg2+ and the four deoxyribonucleoside triphosphates and partially stimulated by ATP.