The Oct-3/4 is a transcriptional factor expressed rather specifically in inner cell mass and embryonic ectoderm during early embryogenesis in mammals. During the course of characterizing the regulatory element of embryonic transcriptional coactivator UTF1 which is one of the downstream genes of Oct-3/4, we have found that Oct-3/4, but not other Octamer factors, shows unique DNA binding specificity, being able to bind to variant Octamer sequence (5'-A (C) under bar TAGCAT-3') present in UTF1 regulatory element. Here, we have determined the DNA binding property of Oct-3/4 in a systematic manner using all possible one point mutants of Octamer sequence as well as completely random sequences. These analyses led to the identification of nine distinct nucleotide sequences including the one (5'-A (C) under bar TAGCAT-3') present in UTF1 regulatory element which serves rather as specific binding site for the Oct-3/4. Moreover, we show that, at least, one of the newly identified sequences, 5'-AT (C) under bar AGCAT-3', exhibits a transcriptional stimulating activity equivalent to that of the variant Octamer sequence present in the UTF1 regulatory. region in embryonic stem cells in a pluripotent-state specific manner. (C) 2003 Elsevier Science (USA). All rights reserved.