The occurrence and localization of molecular components of pro-opiocortin in the pituitary and brain of two species of adult lamprey, Petromyzon marinus and Entosphenus tridentatus, were studied immunocytochemically using antisera generated against human pro-γ-MSH (N-terminal fragment 1Trp to 71Gly of pro-opiocortin), porcine ACTH, α-MSH, human β-endorphin, γ-endorphin, and methionine enkephalin. (1) In both species of lamprey most cells of the rostral pars distalis and some cells of the caudal pars distalis contained Met-enkephalin-like immunoreactivity. Some of these cells also contained γ-endorphin-like immunoreactivity. After preabsorbing the antisera with their corresponding antigens or related peptides, the Met-enkephalin/γ-endorphin-like material was found to be related to Met-enkephalin, but not identical with either Met-enkephalin or γ-endorphin. However, results of anti-pro-γ-MSH, anti-ACTH, anti-α-MSH, or anti-β-endorphin were consistently negative in the pars distalis of both lamprey species. (2) Immunoreaction to anti-Met-enkephalin was found in some cells of the pars intermedia in both species of lamprey. Although the positive reaction had been eliminated by preabsorption with synthetic Met-enkephalin, the diffuseness of the positive stain in the pars intermedia cells resembled an artifactual cross-reaction rather than a specific reaction. In P. marinus, but not in E. tridentatus, similar inconsistent and questionable immunoreactions corresponding to ACTH and α-MSH also occurred in some pars intermedia cells. Results of other antisera (anti-pro-γ-MSH, anti-β-endorphin, or anti-γ-endorphin) were consistently negative in the pars intermedia of both lamprey species. (3) In both species of lamprey β-endorphin-like material was found in the hypothalamus. In E. tridentatus only Met-enkephalin-like material was observed in the hypothalamus, and these two substances were distributed in different neuronal elements. After application of anti-pro-γ-MSH, anti-ACTH, anti-α-MSH, or anti-γ-endorphin, no positive reaction was found in the brain of either species of lamprey. These findings suggest that if a pro-opiocortin-related prohormone exists in the lamprey, it may be chemically different from those of more advanced vertebrates, and it clearly differs in distribution between the brain and parts of the pituitary gland.