Proliferation of rat 3Y1 cells was found to be specifically blocked by trichostatin A (TSA) at two distinct stages in the cell cycle. The first block occurred in the early G1 phase at least 9 h before the G1/S boundary, whereas the second occurred during the G2 phase. When TSA-arrested cells at the G2 phase were released from the inhibition, the cells with 4C DNA entered a new S phase without passage through the M phase, resulting in the formation of proliferative tetraploid cells. The removal of TSA induced a rapid transient increase in the transcription of c-fos and the cells required 15 h to enter the S phase after release. These results suggest that the cells arrested with TSA are quiescent (G0).