The role of protein kinase C (PKC) activation in central sensitization has been demonstrated in previous studies. Enhanced activity of nociceptive dorsal horn neurons induced by peripheral noxious stimuli, such as intradermal injection of capsaicin or mustard oil, could be prevented by PKC inhibitors. Furthermore, a PKC inhibitor selectively blocked thermal hyperalgesia induced by intrathecal application of substance P (SP). This study was designed to determine if PKCƒÑ is activated in the rat dorsal horn in response to intrathecal substance P (SP) injection. Colocalization of NK-1 receptors and phosphorylated-PKCƒÑ (pPKCƒÑ) was observed in the superficial layers of the dorsal horn in L4-L5 segments. Spinothalamic tract (STT) cells were labeled by microinjections of the retrograde tracer fluorogold (FG) into the right thalamus, including the ventral-posterior lateral (VPL) nucleus. We found co-expression of NK-1 receptors and pPKCƒÑ in STT cells in both the superficial and deep layers of the dorsal horn. Immunohistochemical results also showed a significant increase of pPKCƒÑ immunoreactivity in the superficial dorsal horn after SP injection (15 £gl, 20.2 £gg), compared with the control group. In the electrophysiological study, pretreatment with an inhibitor of PKC, NPC15437, blocked the sensitization effect of SP on nociceptive dorsal horn neurons. In summary, our study has indicated that activation of PKC is an important component of NK-1 receptor signaling pathways in the dorsal horn and in the STT neurons.