Abstract The product of the retinoblastoma susceptibility gene (pRB) exerts its growth-regulatory effects during the G 1 phase of the cell cycle, where all pRB present has been assumed to be in the underphosphorylated form. We demonstrate here that pRB is underphosphorylated and firmly bound in the nucleus only in early G 1 phase. All G 0 cells contain bound, underphosphorylated pRB. The duration of the cell cycle and of the G 1 phase seems to be determined by the time during which pRB is underphosphorylated and bound in the nucleus. The observed time lag between the phosphorylation and release of pRB in the G 1 phase and entry into S phase was 6.5 h and independent of the G 1 transit time. The data suggest that pRB is not directly involved in initiation of DNA replication.