T-Antigen was partially purified from nuclei of cells transformed by simian virus 40 (SV 40). When nuclei isolated from either rat liver or quiescent hamster cells were preincubated with T-antigen preparations, there was a marked stimulation of RNA synthesis in an in vitro assay, up to 150% above control levels. The stimulation of RNA synthesis was inhibited by hamster antiserum against T-antigen but not by normal hamster serum. When the T-antigen preparations were fractionated on glycerol gradients, the fractions containing complement-fixing activity with antiserum to T-antigen also had the highest stimulatory activity on nuclear RNA synthesis. T-Antigen was also partially purified from nuclei of cells transformed by a temperature-sensitive A mutant of SV40. When preincubated up to 2 hr at 50 degrees, the T-antigen preparation from these temperature-sensitive A mutants was rapidly inactivated, in terms of both complement-fixing activity and ability to stimulate RNA synthesis in isolated rat liver nuclei. Under the same conditions of preincubation, T-antigen preparations from cells transformed by wild-type SV40 maintained their complement-fixing activity and ability to stimulate RNA synthesis. These results suggest that the biological action of T-antigen may be exerted at the level of transcription.