The opioid peptide dynorphin is important for the regulation of neuronal activity in the spinal cord. Because dynorphin is produced by neurons throughout the neuraxis, there are many putative sources for spinal dynorphin fibers, in addition to those originating from spinal cord neurons. Using a sensitive double-labeling technique combining in situ hybridization and tract tracing, the present study demonstrates that the paraventricular hypothalamic nucleus (PVH) of adult naïve male Sprague-Dawley rats contains large numbers of dynorphin mRNA-producing cells with projections to the spinal cord. Thus, more than 40% of the spinally projecting neurons in PVH were found to express dynorphin mRNA. This novel finding suggests that the PVH is a major source of spinal dynorphin that may be of importance for the processing of pain and visceral information.