Decreased affinity of the retinoblastoma protein (RB) for the nuclear compartment has been correlated with cell cycle-dependent phosphorylation of the RB protein during the G1/S phase of the cell cycle. We examined the effects of microinjected protein-serine/threonine phosphatases types 1 (PP1) and 2A (PP2A) on nuclear association of RB monitored as the resistance of RB to extraction at the G1/S transition. Microinjection of PP1 into either the nucleus or the cytoplasm of cells synchronized in G1 increased the amount of RB that was resistant to extraction from the nucleus. Microinjection of PP2A, however, required direct injection into the nucleus to generate this effect. In addition, we found that nuclear injection of only the PP2A catalytic subunit (PP2AC) and not the complex containing the A and C subunits inhibited RB extraction. Microinjection of either PP1 or PP2A and the resultant increased affinity of RB for the nucleus corresponded with the inhibition of cell cycle progression into S phase. Injection of either phosphatase into cells that had entered S phase did not block DNA synthesis, suggesting that the effect of the injected phosphatases on cell cycle arrest was specific. In vitro biochemical studies with purified PP1 and PP2A showed that intact RB protein phosphorylated by cdc2 kinase served as a substrate for both protein phosphatases. Our results suggest that protein phosphatases may be important regulators of RB function and support the idea that cell cycle progression is regulated by the phosphorylation state of the RB protein.