The larval nervous system of the ascidian Ciona intestinalis exhibits an abstract form of the vertebrate nervous system. The Ci-Galphai1 gene, which encodes a G-protein alpha subunit, is specifically expressed in distinct sets of neurons in C. intestinalis larvae, including papillar neurons of the adhesive organ, ocellus photoreceptor cells, and cholinergic and GABAergic neurons in the central nervous system (CNS). A GFP reporter gene driven by the 4.2-kb 5' flanking region of Ci-Galphai1 recapitulated the endogenous gene expression patterns. Comparative genomic analysis of the Galphai1 gene between C. intestinalis and Ciona savignyi identified an 87-bp highly conserved non-coding sequence located between -3176 and -3090 bp upstream of the gene. Deletion of this conserved upstream sequence resulted in the complete loss of reporter expression in the central nervous system, while reporter expression in the adhesive organ and mesenchyme cells remained unaffected. The conserved upstream sequence can activate gene expression from basal promoters in the brain vesicle, although it requires additional cis-regulatory sequences to fully activate the CNS-specific gene expression. These results suggest that different types of central neurons share a common transcriptional activation mechanism that is different from that of papillar neurons.