The murine Nanog gene, a member of the homeobox family of DNA binding transcription factors, has been shown recently to maintain pluripotency of embryonic stem cells. We have used a sequence homology and expression screen to identify and clone the mouse and human Nanog genes and characterized their phylogenetic context and expression patterns. We report here the gene structure and expression patterns of the mouse Nanog gene, the human Nanog and Nanog2 genes, and six processed human Nanog pseudogenes. Mouse Nanog expression is high in undifferentiated embryonic stem cells and is down-regulated during embryonic stem cell differentiation, concomitant with loss of pluripotency. Murine embryonic Nanog expression is detected in the inner cell mass of the blastocyst. After implantation, Nanog is detectable at embryonic day (E) 6 in proximal epiblast in the region of the presumptive primitive streak. Expression extends distally as the streak elongates during gastrulation and remains restricted to epiblast. Nanog RNA is down-regulated in cells ingressing through the streak to form mesoderm and definitive endoderm. Nanog expression also marks the pluripotent germ cells of the nascent gonad at E11.5-E12.5 and is highly expressed in germ cell tumour and teratoma-derived cell lines. Reverse transcriptase-polymerase chain reaction analysis detected mouse Nanog expression at low levels in several adult tissues. The human Nanog genes are expressed in embryonic stem cells and down-regulated in all adult tissues and differentiated cell lines examined. High levels of human Nanog expression were detected by Northern analysis in the undifferentiated N-Tera embryonal carcinoma cell line. The conservation in gene sequence, structure, and expression of mouse and human Nanog and Nanog2 genes may reflect a common role in the maintenance of pluripotency in both species.