Embryonic stem (ES) cells can self-renew maintaining the undifferentiated state. Self renewal requires many factors such as Oct4, Sox2, FoxD3, and Nanog. NF- is a transcription factor involved in many biological activities. Expression and activity of NF- increase upon differentiation of ES cells. Reportedly, Nanog protein directly binds to NF- protein and inhibits its activity in ES cells. Here, we found a potential binding site of NF- in the human Nanog promoter and postulated that NF- protein may regulate expression of the Nanog gene. We used human embryonic carcinoma (EC) cells as a model system of ES cells and confirmed decrease of Nanog and increase of NF- upon differentiation induced by retinoic acid. Although deletion analysis on the DNA fragment including NF- binding site suggested involvement of NF- in the negative regulation of the promoter, site-directed mutation of NF- binding site had no effect on the Nanog promoter activity. Furthermore, no direct association of NF- with the Nanog promoter was detected during differentiation. Therefore, we conclude that NF- protein may not be involved in transcriptional regulation of Nanog gene expression in EC cells and possibly in ES cells.