Protein phosphatase 2A (PP2A) is a family of multimeric protein phosphatases containing distinct regulatory subunits. The regulatory subunits target the core enzyme to individual phosphoprotein substrates. The PR70 subunit is a member of the PPP2R3/PR72 family of PP2A regulatory subunits. PR70 interacts with the Cdc6 protein, which is involved in the initiation of DNA replication. PR70 contains two EF-hand calcium binding motifs. Calcium binding to PR70 enhances the interaction with the core enzyme. The interaction of PR70 with Cdc6 is not affected by calcium, but calcium causes enhanced recruitment of the core dimer to Cdc6. Cdc6 is stabilized during the G1 phase of the cell cycle by phosphorylation of its N-terminal domain by cyclin E-CDK2, which inhibits degradation mediated by the APC/Ccdh1 ubiquitin ligase. Overexpression and RNA interference studies showed that PR70 can regulate the stability of Cdc6 in a phosphorylation-dependent manner. The data suggest that targeting of PP2A by the PR70 subunit is involved in regulating the initiation of DNA replication.