Transcription factor AREB6 has a unique structure composed of two zinc-finger clusters in N- and C-terminal regions, and one homeodomain in the middle. AREB6 has been known to regulate the expression of the Na, K-ATPase alpha 1 subunit, interleukin 2 and delta-crystallin genes. We determined the optimal binding sites for the N-terminal zinc-finger cluster as GTCACCTGT or TGCACCTGT and for the C-terminal zinc-finger cluster as C/TACCTG/TT by the CASTing method (cyclic amplification and selection of targets). The additional consensus sequence GTTTC/G, in conjunction with the CACCTGT sequence, was selected by the second CASTing for the entire coding region. The N-terminal zinc-finger cluster binds to DNA strongly when the DNA has GTTTC/G in conjunction with the CACCTGT sequence. The homeodomain had no specific DNA binding activity but was found to interact with the N-terminal zinc-finger cluster. Analyses of zinc-finger mutation proteins revealed that the contribution to DNA binding of each N-terminal zinc-finger motif is altered depending on the presence of the additional consensus. Transient transfection assays showed that AREB6 repressed the human 70-kDa heat-shock gene promoter harboring the CACCTGT sequence together with the additional consensus, and that AREB6 activated the promoter harboring the CACCTGT sequence without the additional consensus. These results suggest that AREB6 has multiple conformational states, leading to positive and negative regulations of gene transcription.