Heterotrimeric G proteins play crucial roles as mediators of signaling by many extracellular stimuli. The receptors that activate G proteins constitute the largest and most diverse family of cell surface molecules involved in signal transmission of metazoan cells. To investigate G protein signaling in the central nervous system (CNS) of chordates, we isolated cDNA fragments encoding five different G protein alpha subunits (CiGalpha(x), CiGalpha(q), CiGalpha(i1a), CiGalpha(i1b), and CiGalpha(i2)) from larvae of the ascidian, a simple chordate, Ciona intestinalis. In situ hybridization analysis revealed that each isoform had distinct patterns of spatial distribution in embryos. Among them, CiGalpha(i1a) and CiG alpha(i1b) mRNAs were specifically expressed in the CNS of the larva, whereas CiGalpha(q) transcripts were expressed in small parts of the trunk epidermis and the tip of the tail, but not in the CNS. The CiGalpha(x) expression was widely observed throughout the trunk and tail of the embryos, and the signals were stronger in the epidermis, mesenchyme, and tail muscle cells. Comparison of cDNA sequences and the exon-intron organization indicate that CiGalpha(i1a) and CiGalpha(i1b) are produced by alternative splicing of transcripts from a single gene, CiGalpha(i1). In the cleavage and gastrula stages, transcripts of CiGalpha(i1) were widely distributed in embryos, and the expression then became restricted to the CNS of tailbud embryos and larvae. An exhaustive search has failed to find transducin-type alpha subunits in C. intestinalis. Since CiGalpha(i1) is expressed in the ocellus, CiGalpha(i1) may mediate signals from Ci-opsin1, a visual pigment of the ocellus photoreceptor cells.