The gnathostome (jawed vertebrates) classical pituitary glycoprotein hormones, FSH, LH, and TSH, consist of a common -subunit (GpA1) and unique -subunits (Gp 1, -2, and -3), whereas a recently identified pituitary glycoprotein hormone, thyrostimulin, consists of GpA2 and GpB5. This paper reports the identification, expression, and function of an ancestral, nonclassical, pituitary heterodimeric glycoprotein hormone (GpH) consisting of the thyrostimulin A2 subunit with the classical -subunit in the sea lamprey, Petromyzon marinus, a jawless basal vertebrate. Lamprey (l) GpA2, and lGpH were shown to form a heterodimer by coimmunoprecipitation of lGpA2 with FLAG-tagged lGpH after the overexpression in transiently transfected COS7 cells using a bipromoter vector. Dual-label fluorescent in situ hybridization and immunohistochemistry showed the coexpression of individual subunits in the proximal pars distalis of the pituitary. GnRH-III (1 ) significantly increased the expression of lGpH and lGpA2 in in vitro pituitary culture. Recombinant lampreyGpHwas constructed by tethering theNterminal of lGpA2 to the C terminal of lGpH with a linker region composed of six histidine residues followed by three glycine-serine repeats. This recombinant lamprey GpH activated the lamprey glycoprotein hormone receptor I as measured by increased cAMP/luciferase activity. These data are the first to demonstrate a functional, unique glycoprotein heterodimer that is not found in any other vertebrate. These data suggest an intermediate stage of the structure-function of the gonadotropin/thyroid-stimulating hormone in a basal vertebrate, leading to the emergence of the highly specialized gonadotropin hormones and thyroid stimulating hormones in gnathostomes.