FOXO4 transcription factor, also referred to AFX, contains three putative phosphorylation motif sites for protein kinase B (PKB), Thr32, Ser197, and Ser262, and it is proposed that phosphorylated FOXO4 stays in the cytosol and is imported to the nucleus through dephosphorylation to induce target gene expression. These three sites were revealed to be phosphorylated by PKB in vitro on phosphopeptide analysis, and in cultured cells on immunoblotting with phosphorylation-site specific antibodies. The mutants with either Thr32 or Ser197 replaced by Ala were found mostly in the nuclear but not the cytosol fraction, and treatment with platelet-derived growth factor did not change their distributions in the cells. FOXO4 proteins mutated at these two sites showed 3- to 5-fold higher transcriptional activity than that of the wild type. In contrast, the replacement of Ser262 did not alter the localization or transcriptional activity. These results indicate that phosphorylation at Thr32 and Ser197 is indispensable, whereas that at Ser262 is not critical, for regulation of the nuclear localization and transcriptional activity of FOXO4. These properties are similar to those of FOXO1 and FOXO3, and thus FOXO transcription factors seem to be regulated through a common mechanism by PKB in the growth factor signaling pathway.