The cystine-knot glycoprotein hormone α (GPA) family regulates gonadal and thyroid functions in vertebrates. Little is known concerning GPA family members in primitive chordates. A previous genomic analysis revealed the presence of two genes homologous to the thyrostimulin α subunit (GPA2) in an amphioxus (Branchiostoma floridae); however only one GPA2 homolog contained both the cystine-knot structure and N-glycosylation site characteristic of family members. Gene-specific PCR was used to obtain the cDNA and genomic sequences of the GPA2 homolog of the amphioxus Branchiostoma belcheri. Whole-mount in situ hybridization revealed GPA2 mRNA expression in the anterior part of the nerve cord and on the left side of the central canal. Because amphioxus possesses only one true GPA2 homolog, while vertebrates possess two glycoprotein hormone α subunits (thyrostimulin α, or GPA2, and the common α subunit of gonadal and thyroid glycoprotein hormones, GPA1), our results suggest that GPA1 was acquired later in the vertebrate lineage through gene duplication.