The use of nuclear steroid receptors as ligand-activated transcription factors is a critical event in vertebrate evolution. It is believed that nuclear steroid receptors arose at or before the vertebrate radiation, except for an androgen receptor (Ar) that evolved only in the gnathostome line. We report an androgen-Ar complex in the male sea lamprey (Petromyzon marinus), an extant jawless vertebrate. The androgen with the highest affinity is not testosterone, but its direct precursor, androstenedione (Ad). To establish that the binding moiety in lamprey testis is a receptor—and not an “androgen-binding protein”—we have shown that it can be extracted from the nucleus as well as the cytosol, that the Ad-receptor complex binds to DNA, and that the receptor is approximately twice the size of an androgen-binding protein extracted from the Atlantic salmon testis. The capacity (and high affinity) of binding of the lamprey Ar is such that much of the Ad present in male lampreys becomes sequestered within the testis (as opposed to circulating in the plasma). Concentrations of Ad (but not of testosterone) in plasma and testis tissue are upregulated by injection of lamprey GnRH. Implantation of male lampreys with exogenous Ad significantly accelerates the development of the testis and growth of at least one secondary male characteristic. It appears that all classes of steroid hormones have contributed to the evolution of the regulatory complexity of steroid receptors found in modern vertebrates.