The present study has characterized gonadotropic releasing hormone (GnRH)-like molecules in the brains of representatives of the two southern hemisphere families of lampreys, Geotriidae and Mordaciidae. Chromatographic and immunocytochemical evidence showed that the brains of Geotria australis and Mordacia mordax contain two forms of GnRH-like molecules. These two forms correspond to lamprey GnRH-I and -III, which were first sequenced from the brain of the anadromous sea lamprey Petromyzon marinus, a representative of the family Petromyzontidae that is found only in the northern hemisphere. In chromatographic studies (HPLC) using lamprey GnRH-I and -III antiserum, two early eluting GnRH forms coeluted with synthetic lamprey GnRH-I and -III standards. Our studies thus indicate that, despite their apparently long period of separation, the three families of extant lampreys have each retained both of the lamprey GnRH (-I and -III forms) molecules. Moreover, immunocytochemical localization of lamprey GnRH indicated that the pattern of its distribution in the adult brain of at least one of these southern hemisphere lampreys (G. australis) is similar to that previously described for P. marinus. Distribution of GnRH in the brain of larval G. australis was not as extensive as that in larval P. marinus, which may account for the later gonadal development in the former species. The fact that lamprey GnRH-I and -III are the dominant GnRH forms in all three families of lampreys implies that these neurohormones have an ancient origin.