Antibodies immobilized onto the outer surface of glass microelectrodes were used to measure and localize substance P (SP) release in the spinal cords of anaesthetized rats. Utilizing a C-terminally directed antibody, significant levels of SP were not found in the lumbar spinal cord in the absence of peripheral noxious stimulation. Following noxious heating or pinch of the ipsilateral hind paw or electrical stimulation of the ipsilateral tibial nerve at C-fibre strength, significant amounts of released SP were detected. This noxious stimulus-evoked release of SP was primarily in the region of the substantia gelatinosa. In conclusion, the antibody microprobe technique can be employed to focally detect the release of neuropeptide in vivo, even in structures as small as rat spinal cord. The technique reveals that SP release in the rat follows broadly the same pattern as that previously reported in the cat.