To examine the presence and distribution of Six family gene products in a variety of tissues at various developmental stages and in various cell types, we prepared specific antibodies against recombinant Six gene products. The distribution of Six2 and Six4 was examined by immunostaining in the developing mouse embryo. Production of Six2 was detected at E8.5 mainly in the mesenchyme, while Six4 was present in nuclei of neuronal cells in the peripheral region of the mantle layer of developing brain and spinal cord and in various ganglia at E10.5 and E11.5. Specific DNA binding activities of the Six proteins were analyzed by gel retardation super-shift assays using nuclear extracts from different rat tissues and cell lines. Six5 was the dominant isoform observed in the adult kidney, liver and lung but not in the brain. Six4 was not detected in all tested adult tissues, however, it was present in embryonic (FD21) lung nuclear extracts. In contrast, Six4 was detected in a variety of cultured cell lines, including HeLa, 3T3, MDCK and C2C12. Our results suggest that Six4 plays a specific role in the differentiation or maturation of neuronal cells, while Six5 is an adult type Six gene isoform product and is distributed in the kidney, liver and lung.