A novel member of the winged helix (formerly HNF-3/Forkhead) transcriptional regulatory family, termed Genesis, was isolated and characterized. Putative translation of the complete cDNA revealed the winged helix DNA binding domain to be centrally located within the protein, with regions on either side that contain known transcriptional regulatory motifs. Extensive Northern analysis of Genesis found that the message was exclusively expressed in embryonic stem cells or their malignant equivalent, embryonal carcinoma cells. The Genesis transcript was down-regulated when these cells were stimulated to differentiate. DNA sequences that Genesis protein would interact with were characterized and were found to contain a consensus similar to that found in an embryonic stem cell enhancer sequence. Co-transfection experiments revealed that Genesis is a transcriptional repressor. Genesis mapped to mouse chromosome 4 in a region syntenic with human chromosome 1p31, a site of nonrandom abnormalities in germ cell neoplasia, neuroblastoma, and acute lymphoblastic leukemia. Genesis is a candidate for regulating the phenotype of normal or malignant embryonic stem cells.