Three peptides containing the melanotropin (MSH)-core sequence, YXMXHFRWG, have been isolated recently from the pituitary glands of adult sea lampreys (Petromyzon marinus) and were tentatively assigned as lamprey adrenocorticotropin (ACTH), MSH-A, and MSH-B, respectively. Both MSHs differed significantly from gnathostome MSHs and cannot be assigned as alpha-MSH, beta-MSH, or gamma-MSH. The aim of the present study was to localize these peptides in the lamprey pituitary using antisera generated against synthetic lamprey ACTH1-16, MSH-A, and MSH-B. ACTH-like immunoreactivity was found in most cells of the rostal pars distalis (RPD) and in a few scattered cells of the proximal pars distalis (PPD). MSH-A-like immunoreactivity was found in most cells of the RPD, a few scattered cells of the PPD, and almost all cells of the pars intermedia (PI). MSH-B-like immunoreactivity was found only in the PI, where almost all cells were stained. Thus, the topographic distributions of ACTH and MSHs in the lamprey pituitary were similar to those in gnathostome vertebrates.