The coexistence of the neuropeptides neurotensin and cholecystokinin and the catecholamine-synthesizing enzyme tyrosine hydroxylase within neurons of the ventral mesencephalon was analyzed using an immunofluorescence triple-labeling technique. Virtually all of the neurotensin-positive cell bodies in the ventral tegmental area, medial substantia nigra pars compacta, retrorubral field, and rostral and caudal linear raphe nuclei were found to contain both cholecystokinin and tyrosine hydroxylase immunoreactivities. The degree of colocalization was lower and more variable in other regions including the ventral and central periaqueductal grey matter and dorsal raphe nucleus. It appeared that immunoreactivities for these 3 neuroactive substances were not contained within the same axonal-like fibers and terminals in the ventral midbrain. These results demonstrate that a subpopulation of dopaminergic neurons, which presumably comprise part of the ascending mesotelencephalic system, contains the two peptides neurotensin and cholecystokinin. Thus, the data suggest a morphological basis for some of the reported functional interactions of these 3 putative neurotransmitters/neuromodulators within this system.