We have investigated the effects of intermolecular disulfide crosslinking and temperature-dependent insolubilization of nuclear proteins in vitro on the association of the polyoma large T antigen with the nuclear matrix in polyomavirus-infected mouse 3T6 cells. Nuclear matrices, prepared from polyomavirus-infected 3T6 cells by sequential extraction of isolated nuclei with 1% Triton X-100 (Triton wash), DNase I, and 2 M NaCl (high salt extract) at 4 degrees C, represented 18% of total nuclear protein. Incubation of nuclei with 1 mM sodium tetrathionate (NaTT) to induce disulfide crosslinks or at 37 degrees C to induce temperature-dependent insolubilization prior to extraction, transferred an additional 9-18% of the nuclear protein from the high salt extract to the nuclear matrix. This additional protein represented primarily an increased recovery of the same nuclear protein subset present in nuclear matrices prepared from untreated nuclei. Major constituents of chromatin including histones, hnRNP core proteins, and 98% of nuclear DNA were removed in the high salt extract following either incubation. Polyoma large T antigen was quantified in subcellular fractions by immunoblotting with rat anti-T ascites. Approximately 60-70% of the T antigen was retained in nuclei isolated in isotonic sucrose buffer at pH 7.2. Most (greater than 95%) of the T antigen retained in untreated nuclei was extracted by DNase-high salt treatment. Incubation at 37 degrees C or with NaTT transferred most (greater than 95%) of the T antigen to the nuclear matrix. T antigen solubilized from NaTT-treated matrices with 1% SDS sedimented on sucrose gradients as a large (50-S) complex. These complexes, isolated by immunoprecipitation with anti-T sera, were dissociated by reduction with 2-mercaptoethanol, and SDS-PAGE analysis revealed that T antigen was crosslinked in stoichiometric amounts to several host proteins: 150, 129, 72, and 70 kDa. These host proteins were not present in anti-T immunoprecipitates of solubilized nuclear matrices prepared from iodoacetamide-treated cells. Our results suggest that the majority of polyomavirus large T antigen in infected cells is localized to a specific subnuclear domain which is distinct from the bulk chromatin and is closely associated with the nuclear matrix.