One of the most common molecular changes in cancer cells is the overexpression of fatty acid synthase (FAS), a key metabolic enzyme catalyzing the terminal steps in the synthesis of long chain saturated fatty acids. As part of our efforts to elucidate the mechanisms responsible for FAS overexpression, we have addressed the question whether overexpression of FAS may be linked to the frequently observed inactivation of PTEN and subsequent activation of the phosphatidylinositol 3'-kinase (PI3k) pathway. Using LNCaP prostate cancer cells as an experimental paradigm of FAS-overexpressing PTEN-null cancer cells, we demonstrate that LY294002, an inhibitor of the PI3k pathway causes a dramatic decrease in FAS protein expression. Smaller but still substantial effects are seen at the FAS mRNA level and at the level of transcriptional activity of FAS promoter-reporter constructs. Consistent with these findings, reintroduction of PTEN results in decreased levels of FAS expression in a manner that is dependent on its lipid phosphatase activity. In support of a role for Akt/protein kinase B as a downstream effector, cotransfection of constitutively active Akt1/protein kinase B alpha abrogates the inhibitory effects of PTEN expression and restores FAS promoter activity. Taken together, these results demonstrate that inactivation of PTEN and subsequent activation of the PI3k/Akt kinase pathway may play an important role in the overexpression of the FAS protein in cancer cells.