Immunohistochemical techniques were employed to investigate the distribution of orexin-A-like immunoreactivity in the bullfrog (Rana catesbeiana) pituitary. Orexin-A-immunoreactive cells were scattered throughout the pars distalis. We found that these cells corresponded to the cells immunostained with antiserum against bullfrog prolactin (fPRL). Immunoelectron microscopic analysis indicated that an orexin-A-like substance coexisted with fPRL within secretory granules. Western blot analysis of bullfrog pituitary extract revealed that anti-human orexin-A antiserum labeled two separate bands which were not labeled with anti-fPRL antiserum. The present study has, for the first time, provided evidence of the intragranular colocalization of orexin-A-like and PRL immunoreactivities in the bullfrog pituitary.