We previously reported that PKA activity is an important determinant of thyroid cell survival. Given the important role of cAMP response element binding protein (CREB) in mediating the transcriptional effects of PKA, we explored whether interference with CREB family members impaired thyroid cell survival. Expression of A-CREB, a dominant negative CREB mutant that inhibits CREB DNA binding activity, induced apoptosis in rat thyroid cells. A-CREB inhibited CRE-regulated gene expression, but failed to alter the expression of bcl-2 family members or of well-characterized inhibitors of apoptosis. To elucidate the mechanism through which impaired CREB function triggered apoptosis, its effects on cell proliferation were examined. Expression of A-CREB inhibited cell number increases, in part due to delayed cell cycle transit. Protracted S phase progression in A-CREB-expressing cells was sufficient to activate a checkpoint response characterized by Chk-1, histone H2A.X and p53 phosphorylation. To determine whether cell cycle progression was required for apoptosis, the effects of p27 overexpression were investigated. Overexpression of p27 prevented cell cycle progression, checkpoint activation and apoptosis in A-CREB-expressing cells. These data reveal a novel mechanism through which interference with CREB abrogates cell survival, through checkpoint activation secondary to cell cycle delay. This study may explain how interference with CREB induces apoptosis in cells where alterations in the expression of pro- and anti-survival genes are not detected.