Ectopic expression of cyclins D1 and E was previously shown to accelerate the G1/S-phase transition, indicating that both classes of G1 cyclin control an event(s) that is rate limiting for entry into S phase. In order to determine whether cyclins D1 and E control the same or two different rate-limiting events, we have created cell lines that express both cyclins in an inducible manner. We show here that ectopic expression of both cyclins E and D1 in the same cell has an additive effect on shortening of the G1 interval relative to expression of any single cyclin. In order to further explore the molecular basis for G1 cyclin action, we used cell lines capable of expressing cyclin D1, E, or both prematurely and measured the effect of cyclin expression in early G1 on phosphorylation of the retinoblastoma susceptibility gene product (pRb). We show here that while premature expression of either cyclin alone advances the G1/S-phase transition to the same extent, premature expression of cyclin D1 leads to immediate appearance of hyperphosphorylated pRb, while premature expression of cyclin E does not. Ectopic expression of both cyclins E and D1 in the same cell has an additive effect on shortening of the G1 interval, while the effect on pRb phosphorylation is similar to the effect of cyclin D1 alone. These results suggest that cyclins E and D1 control two different events, both rate limiting for the G1/S-phase transition, and that pRb phosphorylation might be the rate-limiting event controlled by cyclin D1.