The brain (sensory vesicle) of the ascidian larvae is thought to be homologous to the vertebrate forebrain and midbrain and, thus, is proposed as a simplified model to investigate mechanisms of brain formation in vertebrates. However, the genetic circuitry that governs formation of the sensory vesicle is largely unknown. To address this issue, we investigated the transcriptional regulation of the sensory vesicle-specific gene HrTRP by Hroth, the otx gene of the ascidian Halocynthia roretzi. A 133-bp 5'-flanking region of HrTRP, identified as a promoter that can drive expression of the reporter gene in the sensory vesicle, contains two otx binding consensus sites. When the two otx sites were deleted or mutated, the promoter activity of this region was decreased. Hroth overexpression can transactivate this promoter in an otx site-dependent manner. Transactivation of HrTRP promoter by Hroth overexpression was mimicked by overexpression of Hroth/VP16, which encodes a fusion protein of Hroth and the activator domain of VP16, and is suppressed by coexpression with Hroth/En, which encodes a fusion protein of Hroth and the Engrailed repressor domain. Finally, translational interference of Hroth by a morpholino oligonucleotide resulted in the reduction of HrTRP expression in the ascidian embryos. These results suggest that Hroth acts as a direct activator of HrTRP transcription during sensory vesicle development.