We present evidence that the embryo of the ascidian, Ciona intestinalis, is an easily manipulated system for investigating the establishment of basic chordate tissues and organs. Ciona has a small genome, and simple, well-defined embyronic lineages. Here, we examine the regulatory mechanisms underlying the differentiation of the notochord. Particular efforts center on the regulation of a notochord-specific Ciona Brachyury gene (Ci-Bra). An electroporation method was devised for the efficient incorporation of transgenic DNA into Ciona embryos. This method permitted the identification of a minimal, 434 bp enhancer from the Ci-Bra promoter region that mediates the notochord-restricted expression of both GFP and lacZ reporter genes. This enhancer contains a negative control region that excludes Ci-Bra expression from inappropriate embryonic lineages, including the trunk mesenchyme and tail muscles. Evidence is presented that the enhancer is activated by a regulatory element which is closely related to the recognition sequence of the Suppressor of Hairless transcription factor, thereby raising the possibility that the Notch signaling pathway plays a role in notochord differentiation. We discuss the implications of this analysis with regard to the evolutionary conservation of integrative enhancers, and the subdivision of the axial and paraxial mesoderm in vertebrates.