In ascidian embryos, ectodermal tissues derive from blastomeres in the animal hemisphere. The animal hemisphere-specific gene expression is observed as early as the 16-cell stage. Here, we characterized animal hemisphere-specific enhancers of three genes, Ci-ephrin-Ad, Ci-TGFβ-NA1 and Ci-Fz4. Deletion analyses identified minimal essential elements. Although these elements contained multiple GATA sequences, electrophoretic mobility shift assays revealed that only some of them were strong binding sites for the transcription factor Ci-GATAa. On the other hand, the motif-searching software MEME identified an octamer, GA (T/G) AAGGG, shared by these enhancers. In Ci-ephrin-Ad and Ci-TGFβ-NA1, the octamer was GATAAGGG, which strongly bound Ci-GATAa. The 397-bp upstream region of Ci-ephrin-Ad contained two strong Ci-GATAa-binding sites, one of which was the octamer motif. Mutation in the octamer motif, but not the other Ci-GATAa-binding site, severely affected the enhancer activity. The 204-bp upstream region of Ci-TGFβ-NA1 contained four strong Ci-GATAa-binding sites, including the octamer motif. Mutation only in the octamer motif, leaving the other three Ci-GATAa-binding sites intact, abolished the enhancer activity. These results suggest a crucial role for the octamer motif.