A double-label immunofluorescence technique was used to demonstrate that immunoreactivities for the functionally antagonistic neuropeptides enkephalin and cholecystokinin octapeptide (CCK) are co-localized within individual neurons and processes in discrete areas of rat midbrain and forebrain. Coexistence was most prominent within varicose pericellular axons extending from the periaqueductal gray matter to a field overlying the medial lemniscus, axons and terminal-like puncta in the central medial, paracentral, interanterodorsal and ventral anterior thalamic nuclei, and perikarya and proximal axonal fragments in layers II and III of neo- and allocortex, and in the anterior olfactory nucleus. The former two systems of axons lie in areas of spinothalamic tract termination. These data suggest that some of the antagonism of opioid analgesia by CCK occurs at the synaptic level in nociceptive areas of brain-stem and thalamus where CCK and enkephalin are co-localized and presumably co-released.