Cholecystokinin (CCK) receptor binding was measured in rodent and primate brain and spinal cord using¹²⁵I-Bolton Hunter CCK-8 (¹²⁵I-BH-CCK) and the selective non-peptide CCK antagonists MK-329 and L-365,260. In homogenate binding studies, L-365,260 displayed nanomolar affinity for CCK-B receptors in the cerebral cortex of several species including man (pIC₅₀ ∼ 8.2) but showed low affinity for CCK-A receptors in the rat pancreas (pIC₅₀ ≅ 6.3). By contrast, the CCK-A antagonist MK-329 showed the reverse selectivity (cortex:pIC₅₀ ≅ 6.9, pancreas:pIC₅₀ ≅ 9.6). In autoradiographs of rat and monkey brain,¹²⁵I-BH-CCK binding was localized regionally with high levels being detected in the cerebral cortex, basal ganglia and some mid- and hindbrain nuclei. Specific¹²⁵I-BH-CCK binding was also localized to the substantia gelatinosa of the rat, monkey and human spinal cord. L-365,260 inhibited binding to most areas of the brain, but in the rat medial nucleus tractus solitarii and the monkey nucleus tractus solitarii, dorsomedial nucleus and infundibular hypothalamic nuclei together with the dorsomedial aspects of the caudate nucleus, where CCK-A sites are present, L-365,260 failed to displace all¹²⁵I-BH-CCK binding. In the primate spinal cord, L-365,260 was a relatively weak inhibitor of¹²⁵I-BH-CCK binding (pIC₅₀ ≅ 6.0) whereas MK-329 showed high affinity for the CCK-A sites present there (pIC₅₀ ≅ 9.6). In contrast, in the rat L-365,260 (pIC₅₀ ≅ 7.9) showed higher affinity than MK-329 (pIC₅₀ = 7.2) for binding sites in the dorsal horn indicating that CCK-B receptors predominate in the spinal cord of this species. It is concluded that L-365,260 is a selective ligand with high specificity for CCK-B receptors and may be used in conjunction with its analogue, MK-329, to detect CCK receptor subtypes in the brain and spinal cord.