Insulin inhibits expression of certain liver genes through the phosphoinositol (PI) 3-kinase/Akt pathway. However, whether Akt activity is both necessary and sufficient to mediate these effects remains controversial. The forkhead proteins (Foxo1, Foxo3, and Foxo4, previously known as Fkhr or Afx) are transcriptional enhancers, the activity of which is inhibited by insulin through phosphorylation-dependent translocation and nuclear exclusion. Others and we have previously shown that the forkhead protein Foxo1 is phosphorylated at three different sites: S(253), T(24), and S(316). We have also shown that T(24) fails to be phosphorylated in hepatocytes lacking insulin receptors, and we have suggested that this residue is targeted by a kinase distinct from Akt. In this study, we have further analyzed the ability of Akt to phosphorylate different Foxo1 sites in control and insulin receptor-deficient hepatocytes. Expression of a dominant negative Akt (Akt-AA) in control hepatocytes led to complete inhibition of endogenous Akt, but failed to inhibit Foxo1 T(24) phosphorylation and, consequently, insulin suppression of IGFBP-1 promoter activity. Conversely, expression of a constitutively active Akt (Akt-Myr) in insulin receptor-deficient hepatocytes led to an overall increase in the level of Foxo1 phosphorylation, but failed to induce T(24) and S(316) phosphorylation. These data indicate that the Foxo1 T(24) and S(316) kinases are distinct from Akt, and suggest that the pathways required for insulin regulation of hepatic gene expression diverge downstream of PI 3-kinase.