The intranuclear compartments which contain adenovirus type 5 (Ad5) genomes during productive infection of HeLa cells were determined by high resolution when the rate of viral DNA replication is at its maximum. In situ hybridization was performed with and without denaturation of Ad5 DNA to reveal those portions of both double-stranded and single-stranded viral DNA and of single-stranded viral DNA alone, respectively, which were present at the surface of Lowicryl thin sections. Autoradiography after short pulse incorporation of tritiated thymidine revealed sites of active DNA replication whereas a pulse-chase experiment visualized the migration of the synthesized DNA. Immunocytochemistry determined distribution of the Ad5 72 kDa protein. Viral DNA was contained within multiple, well-delimited, compact fibrillar structures and their immediately surrounding, less well-defined, fibrillo-granular areas of the cell nucleoplasm. The compact fibrillar masses were primarily storage sites of single-stranded viral DNA and its accompanying 72 kDa protein, so they were named ssDNA accumulation sites. They contained intermittent viral replicative activity. Conversely, viral ssDNA and 72 kDa protein were rare in the surrounding fibrillo-granular areas in which Ad5 replication DNA is continuous, so we referred to them as peripheral replicative zones. Well-delimited subnuclear structures induced by viruses, therefore, represent accumulations of specific components of their replicating genomes.