Substance P (SP) plays a central role in the transduction of second messenger signals from primary afferent nociceptive terminals to second-order neurons in the spinal cord. We have tested a recombinant engineered diphtheria toxin/SP fusion protein (DAB389SP) in acute and chronic pain models in the rat. DAB389SP binds to the SP receptor (SPR) and is internalized and kills SPR-expressing cells by blocking cellular protein synthesis. DAB389SP delivery was by intrathecal infusion, of varying duration, at the lumbar level. In the chronic constriction injury model of neuropathic pain a significant reduction in mechanically induced hyperalgesia was obtained. This effect was less marked in an acute carageenan inflammation model. Although other pain characteristics (mechano-allodynia, cold-allodynia, and heat-hyperalgesia) showed some improvement, these were less pronounced. Immunocytochemistry revealed a toxin-induced reduction in lamina I, of SPR and of NMDA NR1 subunit receptor expressing neurons, and of c-Fos, an inducible molecular marker of persistent nociceptive activity. The use of cytotoxic fusion proteins to target specific cell types may be of considerable benefit in the study of nociception and the treatment of chronic pain.