To define the relationship between simian virus 40 (SV40)-specific T-antigen and cell growth and to look for regulatory mechanisms that might control T-antigen synthesis in transformed cells, we studied the expression of T-antigen and the viral transcription in SV40-transformed cells that were exponentially growing or arrested in the G1-phase of the cell cycle. We took advantage of the behavior of two lines of SV40-transformed mouse 3T3 cells (ts SV3T3), which, although transformed by wild-type SV40, are temperature sensitive for the expression of the transformed phenotype. At 32 degrees C, ts SV3T3 cells behave like standard transformants, whereas at 39 degrees C, they become arrested in G1 after reaching saturatio n density or under serum starvation. At 32 degrees C or growing at 39 degrees C, ts SV3T3 were 100% T-antigen positive and contained virus-specific mRNA. However, after G1 arrest at 39 degrees C, most of the cells became T-antigen negative. This seems to be caused by a lack of transcription of the integrated viral DNA, since these cells contain no appreciable amounts of SV40-specific RNA. Induction of proliferation in resting, T-antigen-negative ts SV3T3 cultures results in the reappearance of T-antigen a few hours before the cells enter DNA synthesis. These results suggest that transcription of the viral genome and T-antigen expression in SV40-transformed cells is subjected to a cell cycle control.