The lampreys (family Petromyzontidae) are divided into three subfamilies, the Petromyzontinae in the Northern Hemisphere and the Geotriinae and Mordaciinae in the Southern Hemisphere. We previously found two proopiomelanocortin subtypes, proopiocortin (POC) and proopiomelanotropin (POM) in sea lamprey, Petromyzon marinus (Petromyzontinae). POC encoding adrenocorticotropic hormone (ACTH) and β-endorphin (β-END) is expressed in the pars distalis of the pituitary, while POM encoding melanophore-stimulating hormone (MSH)-A and B together with a diVerent β-END is expressed in the pars intermedia of the pituitary. All these hormonal segments are encoded on the third exon in both POC and POM. Here, we demonstrate the presence of both POC and POM genes in Geotria australis (Geotriinae) and Mordacia mordax (Mordaciinae) by molecular cloning of the third exons with the polymerase chain reaction using genomic DNA or pituitary cDNA. Molecular phylogenetic analysis showed that the POC and POM are distinctly diVerent for the Southern Hemisphere lampreys as they are for P. marinus. Moreover, the relationship of each hormonal segments in POC and POM between Geotria, Mordacia, and Petromyzon is inconsistent. Immunocytochemical studies showed that the distribution of POC and POM in the pituitaries of the Southern Hemisphere lampreys is the same as that in the Northern Hemisphere. Taken together, these Wndings suggest that the duplication event which generated the two genes may have occurred in a common ancestor of the three extant lamprey subfamilies.