We analyzed the subnuclear distribution of the simian virus 40 (SV40) large tumor (large T) antigen during the course of viral infection. Three distinct nuclear subclasses were detected in SV40 lytically infected TC7 cells (large T antigen in the nucleoplasm, at the cellular chromatin, and at the nuclear matrix). During the course of infection the relative subnuclear distribution of large T antigen changed significantly at about the switch from the early to late phase of infection: at early times postinfection, large T antigen was present mainly in the nucleoplasm and at the cellular chromatin, and nuclear-matrix-associated large T antigen was barely detectable. Concomitant with the onset of viral DNA replication, the amount of nuclear-matrix-associated large T antigen increased drastically. During the further course of infection large T antigen accumulated at the cellular chromatin and nuclear matrix, paralleling the increase in viral DNA synthesis. The biological significance of this correlation was corroborated by analysis of cells infected with the SV40 mutant tsA58 at permissive (32 degrees C) and restrictive (39 degrees C) temperatures. tsA58 large T antigen failed to initiate viral DNA replication in infected cells kept at the restrictive temperature and also failed to associate with the cellular chromatin and nuclear matrix. By blocking viral DNA synthesis with aphidicolin, an inhibitor of DNA polymerase alpha, we were able to show that the accumulation of large T antigen at these structures does not result from the binding of large T antigen to viral chromatin but reflects an association with cellular components of the chromatin and nuclear matrix of infected cells.