Otx (otd in Drosophila) is a well-conserved homeobox gene throughout animal phylogeny and commonly expressed in the anterior part of the embryo. In embryos of the ascidian Halocynthia roretzi, Hroth, the otx homologue in this species, is expressed in the endoderm and the sensory vesicle, the anterior part of the larval ascidian central nervous system (CNS), which has been thought to be homologous to vertebrate forebrain and midbrain. The developmental expression pattern of Hroth is very similar to that of vertebrate counterparts, which leads to a possibility that a similar mechanism may exist in the patterning of the CNS between ascidians and vertebrates. To better understand the mechanism, we decided to undertake analysis of the transcriptional regulatory regions of Hroth. We isolated and determined the nucleotide sequence of the 11.4-kbp region upstream of the translation start site of Hroth. We found that Hroth transcripts are modified likely with spliced leader RNA; therefore, we could not determine the transcription start site. However, first, we identified three introns that are unknown with vertebrate otx genes. Second, we found two regions that are capable of functioning as a promoter through deletion analysis, one of which appeared to be an endogenous promoter of Hroth. We analyzed the 5′ upstream region 5402-1473bp, the region between 1473 and 5402 base pairs upstream from the translation start site of Hroth, including the putative endogenous promoter. This region was capable of driving Hroth expression in the sensory vesicle lineage cells as well as some other lineages at the early tail bud stage. Deletion analysis of this region suggested that three regions, 1659-1650bp, 1628-1613bp, and 1542-1473bp are responsible for regulating Hroth expression in the sensory vesicle cells at the tail bud stage. Among these regions, no apparent sequence conservation was observed. The present study has revealed a complex organization of transcription regulatory regions for the ascidian otx. Developmental Dynamics 227:104–113, 2003. © 2003 Wiley-Liss, Inc.