An attempt was made to identify the immunoreactive substance P (SP) released from isolated rat spinal cords using reversed-phase high-performance liquid chromatography (HPLC) combined with radioimmunoassay (RIA) for SP. Soaking the spinal cords of newborn rats in Krebs solution containing 90 mM K+ evoked a release of immunoreactive SP as well as of GABA and glycine in a calcium-dependent manner. Capsaicin also evoked a release of immunoreactive SP but not of GABA and glycine. The immunoreactive SP released from rat spinal cords by high K+ or capsaicin was analyzed by HPLC. A single peak was detected by RIA whose elution position coincided with that of synthetic SP.