The Akt pathway is very important in both development and cancer. Here we show that, expression of Casein Kinase I epsilon (CKI epsilon) causes up-regulation of the Akt pathway despite normal protein expression of the pathway inhibitor phosphate and tensin homologue deleted on chromosome ten (PTEN). Conversely, we show that a CKI epsilon/delta-specific inhibitor can inhibit Akt phosphorylation at both Thr308 and Ser473 and drastically reduce phosphorylation of the Akt target Glycogen Synthase Kinase 3 beta (GSK3 beta). These conclusions were confirmed between MCF7 cells transiently transfected with CKI epsilon and Hs578T cells which already express endogenous CKI epsilon. The results suggest that CKI epsilon is a new positive regulator of the Akt pathway. Here we propose that, rather than inhibiting PTEN function, CKI epsilon positively regulates Akt possibly by inhibiting Protein Phosphatase 2A (PP2A). (c) 2008 Elsevier Inc. All rights reserved.