The parabrachial nucleus (PB) is the major relay for ascending visceral afferent information from the nucleus of the solitary tract to the forebrain. We have recently found that PB in the rat also receives a substantial afferent projection from neurons in the marginal zone of the entire length of the spinal and trigeminal dorsal horn. Immunoreactive perikarya stained with antisera against several neuropeptides--including dynorphin, enkephalins, and substance P--have been identified in the marginal zone. We therefore investigated the chemical specificity of the spinoparabrachial projection by combining fluorescent retrograde tracing with immunofluorescence for substance P, dynorphin A1-17, met-enkephalin, and two enkephalin precursor fragments (proenkephalin 192-203 and peptide E). Following PB injections of fluorescent dyes, about half of the retrogradely labeled neurons in the marginal zone stained with antisera against either dynorphin or enkephalin series peptides. Elution-restaining experiments indicated that the dynorphin- and enkephalin-immunoreactivities were contained within separate populations of marginal zone neurons. We could not identify any substance P-immunoreactive perikarya in the marginal zone, but substance P-immunoreactive fibers were seen in close apposition to retrogradely labeled, opioid-immunoreactive cell bodies and dendrites. These results indicate that the dynorphin- and enkephalin-immunoreactive perikarya in the marginal zone of the dorsal horn represent independent neuronal populations. These opioid-immunoreactive neurons, which are believed to have extensive local collateral connections, are the main source of a long ascending projection to the parabrachial nucleus in the rat. Furthermore, opioid neurons in the marginal zone may receive substance P-immunoreactive primary sensory afferents.