To define the roles of subnuclear structure in SV40 infection, the relative distribution of T-antigen (T-ag) in various subnuclear fractions obtained from both lytically infected and transformed African green monkey kidney cells was determined. Depending on the differential sensitivity of nuclear T-ag to extraction by salt and detergent, nuclear T-ag could be separated into nucleoplasmic T-ag, salt-sensitive T-ag and matrix-bound T-ag subclasses. At least fivefold less matrix-bound T-ag was found in transformed cells than in lytically infected cells. While a cAMP-independent protein kinase was detected in the nuclear matrix, the matrix-bound T-ag (94K) could not be phosphorylated in vitro. The removal of cellular chromosomes by DNase caused changes in the interaction of T-ag with nuclear components. The results suggest that the compartmentalization of nuclear T-ag may be determined by its interaction with host chromosomes.