We used Neurobiotin as a retrograde tract tracer in both larval and adult sea lampreys and observed a number of neuronal brainstem populations (mainly reticular and octaval populations and some diencephalic nuclei) that project to the spinal cord, in agreement with the results of previous tracer studies. We also observed small labeled neurons in the ventral hypothalamus, the mammillary region, and the paratubercular nucleus, nuclei that were not reported as spinal projecting. Notably, most of the labeled cells of the mammillary region and some of the ventral hypothalamus were cerebrospinal fluid-contacting (CSF-c) neurons. Combined tract tracing and immunocytochemistry showed that some of the labeled neurons of the mammillary and paratubercular nuclei were dopamine immunoreactive. In addition, some CSF-c cells were labeled in the caudal rhombencephalon and rostral spinal cord, and many were also dopamine immunoreactive. Results with other tracers (biotinylated dextran amines, horseradish peroxidase, and the carbocyanine dye DiI) also demonstrated that the molecular weight or the molecular nature of the tracer was determinant in revealing diencephalic cells with very thin axons. The results show that descending systems afferent to the spinal cord in lampreys are more varied than previously reported, and reveal a descending projection from CSF-c cells, which is unknown in vertebrates. The present results also reveal the existence of large differences between agnathans and gnathostomes in the organization of the dopaminergic cells that project to the spinal cord.