Abstract The origin of the excitatory amino acids (EAA) aspartate (Asp) and glutamate (Glu) released into the dorsal spinal cord extracellular fluid of rats following intradialysate infusion of substance P (SP) was studied using neonatal capsaicin, dorsal rhizotomy and proximal spinal cord transection. Neonatal capsaicin (50 mg/kg i.p.) had no effect on basal EAA release, but significantly inhibited SP-induced release of both Asp (86%) and Glu (70%). Bilateral dorsal rhizotomy enhanced SP-induced release of Asp (152%) and had no effect on Glu release compared to sham-operated controls. Proximal spinal transection (T 8–9) had no effect on basal or SP-induced release of EAAs compared to sham-operated controls. The ability of neonatal capsaicin to inhibit, and dorsal rhizotomy to potentiate Asp release correlates well with their distinct effects on hyperalgesia and suggests that these manipulations do not produce identical lesions. Neonatal capsacin likely interferes with the normal development of EAA interneurons innervated by SP primary afferent C-fibers. Rhizotomy may result in a compensatory up-regulation of SP receptors on EAA interneurons.