An association between newly synthesized human adenovirus type 5 DNA and the nuclear matrix of infected HeLa cells is described. Adenovirus-infected cells were pulsed labeled with [3H]thymidine late in infection and the nuclear matrix was prepared. After a 1-min pulse more than 95% of the labeled viral DNA was matrix associated and, when compared with total cell DNA, was resistant to DNase I digestion. When the pulse is longer or is followed by a chase period, the viral DNA remains nuclear matrix associated and less nuclease sensitive than bulk cellular DNA. The resistance to nuclease digestion may result from the close association of viral DNA with the nuclear matrix or could be due to a number of viral-specific proteins which are nuclear matrix associated. It is concluded that viral DNA synthesis occurs in association with the nuclear matrix and the newly synthesized DNA remains matrix associated until it is incorporated into a mature virus particle.