The method of transneuronal retrograde transport of the Bartha strain of the swine alpha-herpes virus, pseudorabies virus, was used to identify putative interneurons presynaptic to motoneurons that supply a tail-flick muscle in the rat. We also investigated whether these interneurons also contribute to ascending somatosensory pathways. Two to five days after injection of pseudorabies virus into the left abductor caudae dorsalis muscle, and cholera toxin B into the right somatosensory thalamus and midbrain, rats were perfused and spinal cord sections processed immunohistochemically in a two-step procedure to stain cholera toxin B-immunoreactive cells black and pseudorabies virus-immunoreactive cells brown. At short (two-day) survivals, the first spinal neurons to be pseudorabies virus-immunoreactive were in the ipsilateral abductor caudae dorsalis motoneuron pool (S3-S4) and intermediolateral cell column (T12-L2), with a few (0 to five/section) bilaterally in the intermediate zone and around the central canal (all lumbosacral levels). With longer (three- to four-day) survival, more cells were noted (20-50/section) bilaterally (ipsilateral preponderance) in the dorsal and ventral horns of the lumbosacral cord. Many were in lamina I (marginal layer), while few were in lamina II (substantia gelatinosa). At four- and five-day survivals, the numbers of cells increased (20 to 100/section) bilaterally and now included lamina II. The fact that unilateral rhizotomy at L4-Co1 failed to change the distribution of spinal pseudorabies virus labeling suggests that the labeling was due to retrograde transport via the ventral root. In support, bilateral removal of the lumbar sympathetic ganglia, which receive their preganglionic innervation through the ventral root, reduced pseudorabies virus immunoreactivity throughout the thoracic and rostral lumbar spinal cord. These data indicate that there are (i) direct projections from intermediate and dorsal horn cells to abductor caudae dorsalis motoneurons, and (ii) disynaptic connections from dorsal horn (possibly including lamina II) cells to more ventral last-order interneurons. We also suggest that some lamina II cells are presynaptic to lamina I cells that project directly to abductor caudae dorsalis motoneurons. We observed cholera toxin B-immunoreactive cells (five to 20/section) in the expected locations (contralateral lamina I, deep dorsal horn and intermediate zone; lateral spinal nucleus bilaterally). Double-labeled (i.e. pseudorabies virus- and cholera toxin B-immunoreactive) neurons were only occasionally seen in the lateral spinal nucleus and were absent in the spinal gray matter, indicating that segmental interneurons do not collateralize in long ascending sensory pathways to the midbrain and somatosensory thalamus.