Resting cultures of nonpermissive rat 3Y1 cells were infected with simian and T antigen expression and entry into S phase were examined under various conditions of culture. In the complete absence of serum from the medium or at an extremely high cell density, the cells delayed T antigen expression and entry into S phase, leaving the interval between the two events constant. Results using the viral mutants deleted in the coding region for the small t antigen ruled out the role of this antigen in induction of S phase. From these and other results presented, we conclude that the large T antigen induces S phase with the same efficiency under different conditions of cultures. We also present the evidence that the large T antigen function is required and is sufficient for entry into S phase in the second as well as in the first generation.