The development of substance P-like immunoreactivity (SPLI) was studied in the Xenopus embryonic nervous system in order to determine in which neuronal populations and at what developmental times SPLI is expressed. Although Rohon-Beard neurons initially were thought to be the only substance P-immunoreactive cells in the embryonic frog spinal cord, we have demonstrated that several neuronal phenotypes are immunoreactive. The earliest evidence of SPLI was seen at stage 28 (Nieuwkoop and Faber, '67), at which time only some trigeminal ganglion cells, their axons in the ophthalmic nerve, and axons in the lateral tracts of the hindbrain showed SPLI. In the embryonic brain at stages 29/30, 37/38, and 42, SPLI was seen in the hypothalamus, trigeminal ganglion cells and their peripheral axons, the sensory roots of cranial nerve IX/X, and axons in the hindbrain lateral tracts. At premetamorphic stages, SPLI was found in several populations that are immunoreactive in adult amphibia. In the embryonic spinal cord, Rohon-Beard neurons were labeled consistently with reaction product; there was a rostrocaudal time gradient of immunoreactivity with increasing development. The Rohon-Beard neurons were not immunoreactive at developmental stages in which axonal outgrowth was beginning (stage 21), but were strongly immunoreactive at stages in which target cells had been contacted (stage 29). Several types of interneurons in the spinal cord (as classified by Roberts and Clarke, '82) showed SPLI during embryonic stages. At premetamorphic stages the Rohon-Beard neurons began to disappear and the immunoreactive interneurons were distributed similarly to those reported in the adult. Dorsal root ganglia differentiated during these stages, and at this time some of the neurons belonging to these ganglia exhibited substance P-like immunoreactivity.