The regional distribution of cholecystokinin (CCK) in the rat brain was determined utilizing a radioimmunoassay which detects both gastrin and CCK. CCK concentration is highest in the caudate nucleus (10-14 ng CCK 8 equivalents/mg protein), followed by the cerebral cortex. Within the cerebral cortex, CCK is highest in the cingulate, pyriform, and entorhinal areas. There are substantial CCK concentrations in all other brain regions except pons, medulla and cerebellum. CCK is widely distributed in the hypothalamus, where it is highest in the median eminence and ventromedial nucleus. Considerable CCK-like immunoreactivity is also present in the posterior lobe of the pituitary gland, but is not detectable in anterior and intermediate lobes. Though the antisera used in this study cross-react with gastrin the dominant CCK-like material found in rat brain co-elutes with sulfated CCK 8 and separates from gastrin on Sephadex G-25 and HPLC chromatography.