FOXO1 (FKHR), one of the forkhead family of transcription factors, is involved in proapoptotic signaling. Phosphorylated Akt leads to the inhibition of FOXO1 activity by direct phosphorylation and cytoplasmic sequestration. We investigated the localization of FOXO1 in non-small cell lung cancer (NSCLC) cell lines grown in a variety of media. FOXO1 expression in tissue microarrays of clinical NSCLC samples was analyzed immunohistochemically. Significant correlations between these data were assessed in order to discern the relationship between Akt and apoptosis. While FOXO1 localized to the nucleus in cell lines cultured without FBS, FOXO1 translocated from the nucleus to the cytoplasm in cell lines cultured with fetal bovine serum (FBS). Inhibition of Akt using RNA interference resulted in the accumulation of FOXO1 in A549 and EBC1 cell nuclei, and in accelerated apoptosis induction. In NSCLC cells, where FOXO1 was present in the cytoplasm constitutively, inhibition of Akt led to FOXO1 translocation to the nucleus and initiation of apoptosis. Using immunohistochemistry and immunofluorescence to examine the expression of FOXO1 in 185 surgically resected, paraffin-embedded stage I-IV tumor samples, we observed a significant correspondence between positive staining for FOXO1 in early stage tumors and in tumors without nodal involvement; in contrast, no significant relationship was observed between FOXO1 expression and tumor status, lymphatic invasion, or venous invasion. Moreover, induction of apoptosis was vigorous in NSCLC cells expressing FOXO1. These results suggest that FOXO1 expression is a favorable prognostic factor in NSCLC.