Immunohistochemical techniques were utilized to investigate the distribution and morphology of neurons containing the calcium binding proteins parvalbumin (PV) and calbindin D28k (CaBP) in the superficial layers of rat spinal cord. Most PV-immunoreactive (PV-IR) neurons were restricted to a 25 to 60 microns thick band straddling the border between lamina II and III. Positive somata had long rostrocaudally oriented dendrites confined to narrow sagittally arranged sheets within this band and axons that entered lamina II or the superficial portions of lamina III. Long varicose axons, presumed to originate from these cells, were moderately distributed in Lissauer's tract and lamina II. CaBP-immunoreactive (CaBP-IR) neurons were found within lamina I and throughout lamina II. Large calibre PV-IR and CaBP-IR axons were seen in the dorsal column and the lateral funiculus. Dorsal rhizotomy or neonatal capsaicin treatment appeared to have no effect on PV-IR and CaBP-IR elements in the superficial lumbar dorsal horn. However, dorsal rhizotomy reduced the number of positive axons in the dorsal column and in deeper lamina of the dorsal horn. These results add to the known lamination patterns of the superficial dorsal horn and point to the existence of a lamina defined by PV-positive neurons at the lamina II/III border. These neurons may have electrophysiological characteristics attributed to PV- or CaBP-containing neurons elsewhere in the CNS.