The role of neurokinin-1 (NK-1) receptors in the rostral ventromedial medulla (RVM) was studied using extracellular single-unit recording combined with microiontophoresis. In rats, ON- and OFF-type neurons were identified using noxious heat or mechanical stimuli applied to the tail. Responses evoked by iontophoretic application of N-methyl-D-aspartate (NMDA) were determined before and after intraplantar injection of capsaicin or iontophoretic application of substance P. In OFF cells, capsaicin produced an extended pause in ongoing activity but did not alter the subsequent spontaneous discharge rate or NMDA-evoked responses. In contrast, spontaneous discharge rates of ON cells increased after capsaicin, and their responses to NMDA increased >100% above control values. The increased responses to NMDA after capsaicin were attenuated by iontophoretic application of the selective NK-1 receptor antagonist L-733,060. Similarly to capsaicin, iontophoretic application of the selective NK-1 receptor agonist, [Sar9,Met(O2)11]- substance P (SM-SP), increased the spontaneous discharge rate and NMDA-evoked responses of ON cells by >100% of control values. These effects were antagonized by L-733,060. Immunohistochemical studies showed that a subset of neurons in the RVM labeled NK-1 receptors and that nearly all of these neurons were immunoreactive for the NMDAR1 subunit of the NMDA receptor. These results demonstrate that activation of NK-1 receptors in the RVM enhances responses of ON cells evoked by NMDA. It is suggested that activation of NK-1 receptors in the RVM and the ensuing sensitization of ON cells may contribute to the development of central sensitization and hyperalgesia after tissue injury and inflammation.