Nuclear matrix prepared from mouse 3T6 cells lytically infected with polyoma virus retained significant amounts of the 100K T antigen and intact viral genomes. Bound T antigen was resistant to the extraction by high salt (2 M NaCl), detergent (1% Triton X-100) and exhaustive DNAase treatment. Only conditions sufficient to disrupt the integrity of the matrix itself solubilized the matrix T antigen. During the time period of 16-30 hr after infection, both the accumulation (in microgram) and the incorporation of 35S-methionine into T antigen increased steadily in cell extracts to a peak at 26 hr and then declined. In contrast, the amount of labeled T antigen retained by the matrix was relatively constant over the same time period. Matrix-bound T antigen was more highly phosphorylated and newly synthesized compared with the extractable T antigen. Viral DNA steadily accumulates in nuclei and on the matrix from 18 to 30 hr after infection. The fraction of viral DNA retained by the matrix was greatest early in infection (25% at 16 hr), declining to less than 10% by 24 hr. These data are consistent with the existence of a fixed (and limited) number of sites for T antigen (more highly phosphorylated) on the matrix and implicate the nuclear matrix as a site of viral DNA replication and possibly encapsidation.