The relationship between viral DNA synthesis and virion assembly was studied in adenovirus type 2 infected HEp2 cells. When cells were infected at the restrictive temperature with ts3, an assembly-negative mutant which permits normal viral DNA and protein synthesis, labeled and shifted to the permissive temperature, only de novo synthesized nonradioactive viral DNA was encapsidated. This suggested that only concurrently synthesized DNA is encapsidated. Blocking protein or DNA synthesis with cycloheximide or hydroxyurea after the temperature shift inhibited virus assembly. Therefore efficient virus assembly requires both concurrent protein and DNA synthesis. When DNA synthesis was arrested by shifting ts125 infected cells to the restrictive temperature, protein synthesis continued but assembly was completely blocked. Sucrose gradient sedimentation analysis of nuclear extracts of wt and ts3 infected cells provided evidence in support of a physical coupling between replication complexes and virus assembly complexes. Further evidence of coupling was also shown by preferential pulse labeling of the molecular right end of the genome isolated from reversibly cross-linked assembly intermediate particles. While DNA replication is not dependent on concurrent virion assembly, at least some significant proportion of replication complexes appear to be coupled to and are prerequisite for virion assembly.