A cascade of events is triggered upon the addition of growth factor to quiescent mammalian cells, which ultimately restarts proliferation by inducing the transition from G0/G1 to S-phase. We have studied cyclin D1, a putative G1 cyclin, in normal diploid human fibroblasts. Cyclin D1 accumulated and reached a maximum level before S-phase upon the addition of serum to quiescent cells. The protein was localized to the nucleus, and it disappeared from the nucleus as cells proceeded into S-phase. Microinjection of anti-cyclin D1 antibodies or antisense plasmid prevented cells from entering S-phase, and the kinetics of inhibition showed that cyclin D1 is required at a point in the cell cycle earlier than cyclin A. These results demonstrate that cyclin D1 is a critical target of proliferative signals in G1.