A family of octamer binding proteins is expressed during mouse development. Oct-4 and Oct-6 have been identified as two octamer binding proteins present in embryonal stem cells. Here we report the complementary DNA cloning and characterization of the mouse Oct-6 gene. The protein of 448 amino acids contains a glycine/alanine-rich amino terminal region, a histidine-rich sequence with homology to a region of kininogen associated with clotting, a POU domain and a short proline/histidine-rich carboxy terminal region. Expression of Oct-6 in HeLa cells is sufficient for transcriptional activation from the octamer motif, identifying Oct-6 as a transcription factor. The Oct-6 expression is downregulated upon embryonic stem cell differentiation increasing again during brain development. Expression in brain is present in certain areas of telencephalon, mesencephalon and brain stem with abundant expression in the cortex anlagen and in the developing colliculi. Thus Oct-6 is a new octamer binding transcription factor specifically regulated during mouse development.