During continuous proliferation, cyclin D1 protein is induced to high levels in a Ras-dependent manner as cells progress from S phase to G2 phase. To understand the mechanism of the Ras-dependent cyclin D1 induction, cyclin D1 mRNA levels were determined by quantitative image analysis following fluorescent in situ hybridization. Although a slight increase in mRNA expression levels was detected during the S/G2 transition, this increase could not explain the more robust induction of cyclin D1 protein levels. This suggested the involvement of post-transcriptional regulation as a mechanism of cyclin D1 protein induction. To directly test this hypothesis, the cyclin D1 transcription rate was determined by run-on assays. The transcription rate of cyclin D1 stayed steady during the synchronous transition from S the G2 phase. We further demonstrated that cyclin D1 protein levels could increase during G2 phase in the absence of new mRNA synthesis. alpha-Amanitin, a transcription inhibitor, did not suppress cyclin D1 protein elevation as the cells progressed from S to G2 phase, even though the inhibitor was able to completely block cyclin D1 protein induction during reentry into the cell cycle from quiescence. The half life of cyclin D1 protein was shortest during S phase indicating that a change in protein stability might play a role in post-translational induction of cyclin D1 in G2 phase. These data indicate a fundamental difference in the regulation of cyclin D1 production during continuous cell cycle progression and re-initiation of the cell cycle.