The process of establishing the anterior-posterior axis is an important event in the development of bilateral animals. Otx, which encodes a homeodomain transcription factor, is continuously expressed in the anterior part of the embryo in a wide range of animals. This pattern of expression is thought to be important for the formation of anterior neural structures, but the regulatory mechanism that sustains the expression is not known. Here, using embryos of the ascidian, Halocynthia roretzi, we investigated how the transcription of Otx is maintained in the cells of the anterior neural lineage during embryogenesis. We identified an enhancer region sufficient to mimic the Otx expression pattern from the gastrula to tailbud stages. Several putative transcription factor binding sites that are required for generating the Otx expression pattern were also identified. Distinct sets of sites were required at different developmental stages, suggesting that distinct transcriptional mechanisms regulate Otx transcription in each of the gastrula, neurula and tailbud stages. Along with previous studies on the transcriptional regulatory mechanism of Otx during the pre-gastrula stages, the present results provide the first overview of the mechanism that sustains Otx expression in the anterior neural lineage during ascidian embryogenesis and demonstrate the complexity of a developmental mechanism that maintains Otx transcription.