We have studied the role of the retinoblastoma susceptibility gene product (pRB) in the regulation of cell-cycle progression under extremely hypoxic conditions (< 4 ppm O2). pRB is a nuclear matrix-associated phosphoprotein that normally exerts its growth-regulatory effects during early-G1 phase of the cell cycle, where all pRB present has been assumed to be in the under-phosphorylated form and bound in the nucleus. The effect of hypoxia on pRB nuclear binding and its state of phosphorylation was studied by two methods: (a) two-parametric flow cytometric measurement of pRB versus DNA and (b) Western blotting. Pulse-chase and pulse labeling with BrdUrd was used to record cell-cycle progression under versus after extremely hypoxic conditions. We demonstrate that pRB is dephosphorylated and rebound in the nucleus in more than 90% of cells located in S and G2 under extremely hypoxic conditions. While inhibition of DNA synthesis was instantaneous under hypoxic conditions, dephosphorylation and rebinding to nuclear structures of pRB takes more than 4 h. Within this time span, cells in G2 complete mitosis and divide. The slow dephosphorylation of pRB indicates that pRB is neither associated with the instantaneous inhibition of DNA synthesis nor is it the cause of the oxygen-dependent restriction point located in late-G1. The observed dephosphorylation of pRB is not dependent on functional p53, suggesting that at least one of the mechanisms responsible for the dephosphorylation is due to hypoxic activation of a pRB-specific phosphatase in the absence of p53-dependent inhibition of pRB kinase activity. However, it cannot be ruled out the participation of pRB kinase inhibitors independent of p53 activation. Cells arrested in G1 during prolonged hypoxia resumed cell-cycle progression within 2-->24 h after reoxygenation, while cells arrested in S were unable to reenter cell-cycle progression after reoxygenation. The hypoxia-induced dephosphorylation of pRB was only partly reversible by reoxygenation. Reentry into the cell cycle induced by reoxygenation occurred concomitant with unbinding (hyperphosphorylation) of pRB. Thus, rephosphorylation of pRB seem to be the rate-limiting step for reentry into the cell cycle after reoxygenation. Although pRB seems to play a major role in suppression of cell growth under and following hypoxic stress, other factors seem to be responsible for the immediate hypoxia-induced arrest in late-G1 and S phases.