The colocalization of neurotensin- and cholecystokinin-like immunoreactivities was demonstrated in neurons of the ventral mesencephalon of the rat by using a double-labeling indirect immunofluorescence procedure for the simultaneous detection of two antigens in the same tissue section. Greater than 90% of the neurotensin-positive perikarya distributed throughout the ventral midbrain (primarily located in the ventral tegmental area, medial substantial nigra, and rostral and caudal linear raphe nuclei) were found to also contain cholecystokinin immunoreactivity. Neurons single-labeled for either peptide were also present, with those immunoreactive for cholecystokinin alone far outnumbering those containing only neurotensin. By combining the double-labeling colocalization technique with fluorescence retrograde tracing, some of the forebrain projections of these neurons were determined. Ventral mesencephalic neurons containing both neurotensin and cholecystokinin were found to project to the nucleus accumbens, prefrontal cortex, or amygdala. The present results, combined with those of previous studies, suggest that there are complex heterogeneous subpopulations of presumed dopaminergic ventral mesencephalic neurons which give rise to the ascending mesotelencephalic systems and which may contain both neurotensin and cholecystokinin, either peptide alone, or neither of these two peptides.