Previous studies with simian virus 40-transformed mouse 3T3 cells which are temperature sensitive for the expression of the transformed phenotype (ts SV3T3 cells) have shown that T-antigen expression and viral DNA transcription are under cell cycle control. Using these ts SV3T3 cells, we studied the expression of the viral genome under proliferating and non-proliferating conditions, in the presence and absence of inhibitors of macromolecular synthesis and of the tumor promoter phorbol myristate acetate. ts SV3TE cells which are growth arrested at 39 degrees C by low serum concentration or saturation density accumulated in G1 and did not express T-antigen. When these cells were induced to proliferate, at either 32 or 39 degrees C, T-antigen synthesis preceded the entry of the cells into the S-phase and was not coupled to DNA replication. G1-arrested ts SV3T3 cells were induced to synthesize T-antigen by phorbol myristate acetate treatment, but T-antigen alone was not sufficient to induce cellular DNA synthesis. Isoleucine deprivation arrested growth of ts SV3T3 cells, but these cells, as well as normal 3T3, did not accumulate in G1 and continued to express T-antigen. The temperature-sensitive expression of the transformed phenotype in the ts SV3T3 cells does not appear to be due to a lack of transcription of specific regions of the integrated simian virus 40 genome at 39 degrees C.