Glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and cardiotrophin-1 (CT-1) are the most potent neurotrophic factors for motoneurons, but their fate after retrograde axonal transport is not known. Internalized trophic factors may be degraded, or they may be recycled and transferred to other neurons, similar to the known route of tetanus toxin. We tested whether neonatal rat hypoglossal motoneurons target retrogradely transported trophic factors to synaptic sites on their dendrites within the brainstem and subsequently transfer these trophins across the synaptic cleft to afferent synapses (transsynaptic transcytosis). Motoneurons retrogradely transport from the tongue radiolabeled GDNF, BDNF, and CT-1 as well as tetanus toxin. Quantitative autoradiographic electron microscopy showed that GDNF and BDNF were transported into motoneuron dendrites with labeling densities similar to those of tetanus toxin. Although tetanus toxin accumulated rapidly (within 8 h) at presynaptic sites, GDNF accumulated at synapses more slowly (within 15 h), and CT-1 never associated with synapses. Thus, some retrogradely transported neurotrophic factors are trafficked similarly but not identically to tetanus toxin. Both GDNF and BDNF accumulate at the external (limiting) membrane of multivesicular bodies within proximal dendrites. We conclude that tetanus toxin, GDNF, and BDNF are released from postsynaptic sites and are internalized by afferent presynaptic terminals, thus demonstrating transsynaptic transcytosis. CT-1, however, follows a strict degradation pathway after retrograde transport to the soma. Synaptic and transcytotic trafficking thus are restricted to particular neurotrophic factors such as GDNF and BDNF.