In this study, we show that depletion of Chk1 by small interfering RNA (siRNA) results in failure of reentry to the cell cycle after DNA replication has been stalled by exposure to hydroxyurea (HU). Casein kinase II (CKII) is degraded in these cells in a proteasome-dependent manner, resulting in decreased phosphorylation and PTEN levels. We show that phosphorylation of Chk1 at Ser(317) but not at Ser(345) is required for phosphorylation of PTEN at Thr(383) by CKII, making cell cycle reentry after HU treatment possible. Like Chk1 depletion, loss of PTEN due to siRNA is followed by inability to return to the cell cycle following HU. In Chk1-siRNA cells, reintroduction of wild-type PTEN but not PTEN T383A restores the ability of the cell to reenter the G(2)-M phase of the cell cycle after stalled DNA replication. We conclude that, in response to stalled DNA replication, Chk1 is phosphorylated at Ser(317) by ATR resulting in stabilization of CKII, which in turn leads to phosphorylation of PTEN at Thr(383).