The activity of transcription factor FoxO1 is regulated by phosphorylation-dependent nuclear exclusion and deacetylation-dependent nuclear retention. It is unclear whether and how these two post-translational modifications affect each other. To answer this question, we expressed FoxO1 cDNAs with combined mutations of phosphorylation and acetylation sites in HEK-293 cells and analyzed their subcellular localization patterns. We show that mutations mimicking the acetylated state (KQ series) render FoxO1 more sensitive to Akt-mediated phosphorylation and nuclear exclusion and can reverse the constitutively nuclear localization of phosphorylation-defective FoxO1. Conversely, mutations mimicking the deacetylated state (KR series) promote FoxO1 nuclear retention. Oxidative stress and the Sirt1 activator resveratrol are thought to promote FoxO1 deacetylation and nuclear retention, thus increasing its activity. Accordingly, FoxO1 deacetylation was required for the effect of oxidative stress (induced by H(2)O(2)) to retain FoxO1 in the nucleus. H(2)O(2) also inhibited FoxO1 phosphorylation on Ser-253 and Thr-24, the key insulin-regulated sites, irrespective of its acetylation. In contrast, the effect of resveratrol was independent of FoxO1 acetylation and its phosphorylation on Ser-253 and Thr-24, suggesting that resveratrol acts on FoxO1 in a Sirt1- and Akt-independent manner. The dissociation of deacetylation from dephosphorylation in H(2)O(2)-treated cells indicates that the two modifications can occur independently of each other. It can be envisaged that FoxO1 exists in multiple nuclear forms with distinct activities depending on the balance of acetylation and phosphorylation.