DNA polymerase alpha (pol-alpha) is a heterotetrameric enzyme (p180-p68-p58-p48 in mouse) that is essential for the initiation of chain elongation during DNA replication. The catalytic (p180) and p68 subunits of pol-alpha are phosphorylated by Cdk-cyclin complexes, with p68 being hyperphosphorylated by cyclin-dependent kinases in G(2) phase of the cell cycle. The activity of Cdk2-cyclin A increases during late S phase and peaks in G(2) phase. We have now examined the role of p68 in the interaction between the catalytic subunit of pol-alpha and hyperphosphorylated retinoblastoma protein (ppRb) and in the stimulation of the polymerase activity of pol-alpha by ppRb. With the use of recombinant proteins, we found that nonphosphorylated p68 inhibited the stimulation of pol-alpha activity by ppRb, suggesting that p68 might impede the association of ppRb with p180. Phosphorylation of p68 by Cdk2-cyclin A greatly reduced its inhibitory effect. Immunofluorescence analysis also revealed that ppRb localized at sites of DNA replication specifically in late S phase. These results suggest that Cdk-cyclin A can phosphorylate pol-alpha which may result in a conformational change in pol-alpha facilitating its interaction with and activation by ppRb.