We studied the organization of the dorsal column nucleus (DCN) of larval sea lamprey with immunohistochemical and tract-tracing techniques. Texas red-coupled dextran amine was injected into the spinal cord, which allowed tracing the dorsal column fibers and characterizing the DCN. The dorsal column fibers formed a dense tract coursing adjacent to the dorsal midline of the spinal cord to the caudal rhombencephalon alar plate. In larvae, most spinal cord dorsal cells and spinal ganglion perikarya, and many dorsal column fibers, were calretinin-immunoreactive. We delineated the DCN in the dorsomedial portion of the obex and preobecular alar plate. It consists of a periventricular neuronal cell layer and neurons scattered in the lateral neuropil and receives dorsal column fibers. After immunohistochemistry with antibodies against glutamate, glycine, and GABA numerous immunoreactive perikarya were observed in the DCN. In addition to glutamate-, glycine-, and GABA-immunoreactive processes, serotonin- and dopamine-immunoreactive fibers coursed in the neuropil of this nucleus. A few small calretinin-immunoreactive perikarya were also observed in the DCN. Our results reveal the presence of inhibitory and excitatory transmitters in neurons of the DCN, and suggest that dopamine and serotonin modulate the activity of this nucleus.