Transforming growth factor alpha (TGF alpha) has been immunocytochemically localized in neuronal perikarya throughout the adult rat central nervous system (CNS). In order to determine if any TGF alpha-immunoreactive (TGF alpha-I) cell bodies have long axonal projections, indirect immunofluorescence was used in combination with injections of the fluorescent retrograde tracer, Fluoro-Gold (FG). Both TGF alpha-I and retrogradely transported FG were found within the same neurons in the interpeduncular nucleus (IPN) after bilateral FG injections in the dorsal tegmental nucleus (DTg). Neurons that contain both TGF alpha-I and FG were also found in the raphe magnus, raphe obscurus, raphe pallidus and the gigantocellularis reticular nuclei after FG injections in the upper thoracic spinal cord. Cell bodies double-labeled with TGF alpha-I and FG were found in the dorsal parabrachial nucleus (DPB) following FG injections in the central nucleus of the amygdala (Ce). In addition, Leu-enkephalin immunoreactivity (L-ENK-I) was colocalized with TGF alpha-I in the same projection neurons after the injections described above. These results suggest that cells that contain TGF and the opioid peptide, L-ENK, have long projections in the rat CNS, and that, due to their co-localization within the same neurons, they may exert their effects concomitantly.