Endomorphin-1 (EM1) and endomorphin-2 (EM2) are the selective endogenous ligands for the mu-opioid receptor (MOR). Since EMs-expressing neuronal cell bodies or axonal components have been observed, respectively, in the nucleus tractus solitarii or the parabrachial nuclei, we examined if EMs-expressing neurons in the NTS of the rat might send their axons to the PBN. Immunofluorescent stainings for EM1 or EM2 were combined with retrograde or anterograde tract-tracing method. After injecting tetramethyl rhodamine dextran-amine (TMR) into the parabrachial nuclei of rats, some EM1- or EM2-immunoreactive neurons in the nucleus tractus solitarii were labeled retrogradely with TMR. The majority of the EM1/TMR and EM2/TMR double-labeled neurons were observed in the medial, commissural, and dorsolateral subnuclei of the nucleus tractus solitarii. Following injection of biotinylated dextran amine (BDA) into the medial, commissural, or dorsolateral subnuclei of the nucleus tractus solitarii, EM1- or EM2-immunopositive axons and axon terminals were anterogradely labeled with BDA mainly in the lateral parabrachial nucleus. The present results have indicated that endomorphinergic neurons in the nucleus tractus solitarii project to the parabrachial nuclei. This suggests that EMs released from NTS-PBN projection fibers may bind to MOR on the PBN neurons to be implicated in processing of visceral information within the parabrachial nuclei.