Thymosin-beta4 is a highly conserved actin-binding protein that plays an important role in multiple early developmental events and functions in keeping the adult life in vertebrates. Here a cDNA for a thymosin-beta4 gene was isolated from the amphioxus, Branchiostoma belcheri. A molecular phylogenetic tree constructed from the deduced amino acid sequence of the isolated cDNA indicates that this gene belongs to the thymosin-beta4 subfamily, but it is split at the base of the vertebrate gene clade in evolution. In situ hybridization reveals that the expression is detected in the locations homologous to orthologous genes expressing regions of vertebrate embryos and adults, such as the neural plate, neural tube, paraxial mesoderm, differentiating somites, pharynx and gut, midgut diverticulus, blood vessels and body spaces. These results are interpreted to mean that thymosin-beta4 genes might play a conserved role in the patterning of chordate embryos and functions in adults.