Ascidians are primitive chordates in which a well-organized notochord is formed in the tail of the tadpole larva. The Brachyury (T) gene in the mouse is essential for formation of mesoderm and, in particular, of notochord. We report here the expression of an ascidian homolog (As-T) of the mouse T gene. A cDNA clone for the As-T gene contains a single open reading frame that encodes a polypeptide of 471 amino acids. Although the overall degree of amino acid identity was not very high (39.9%, ascidian/mouse), in the N-terminal half the extent of amino acid identity was 78.5% (ascidian/mouse). The expression of As-T was transient. A single band corresponding to a 2.2 kb mRNA was first detected at the 64-cell stage, and a distinct band was found at the gastrula stage, at which time accumulation of the transcripts was maximal. The transcript was barely detectable at the larval stage and was undetectable in adult tissues. The occurrence of As-T transcripts was restricted to notochord-lineage cells, and no other cell types expressed the As-T gene. In addition, the timing of As-T expression coincided with the time of restriction of developmental fate exclusively to notochord in the notochord-lineage cells. It is likely that the Brachyury gene was present when the ancestors of chordates with a notochord emerged and that the primary function of this gene is to specify notochord cells.