The involvement of cAMP-responsive element-binding protein (CREB) signaling in tissue injury-induced inflammation and hyperalgesia has been characterized by measuring phosphorylation of CREB at serine-133 (CREB Ser133) using a specific antibody. In the unstimulated state, unphosphorylated CREB was observed in most nuclei of spinal neurons except for motor neurons, where only a small portion of neurons were stained. A few dorsal root ganglion (DRG) neurons were also CREB-positive. After a unilateral injection of formalin into the hindpaw, a strong and bilateral phosphorylation of CREB Ser133 was induced, as assessed by both immunohistochemistry and Western blot. PhosphoCREB (pCREB)-positive neurons were found in laminae I, II, V, and X of spinal cord on both sides. CREB phosphorylation was very rapid and reached peak levels within 10 min of formalin treatment, whereas few pCREB-positive neurons were seen in unstimulated spinal cord. The induction of pCREB was predominantly postsynaptic, because only 5% of DRG neurons were labeled after inflammation. In contrast to CREB phosphorylation, the induction of c-Fos expression reached peak levels 2 hr after formalin treatment and c-Fos induction was mainly ipsilateral. Both formalin-evoked CREB phosphorylation and c-Fos expression in the spinal cord were suppressed by pretreatment with the NMDA receptor antagonist MK-801 (3.5 mg/kg, i.p.) or halothane anesthesia. These results suggest that CREB signaling may play a role in the long-term facilitation of spinal cord neurons after hyperalgesia. Furthermore, our results indicate that CREB phosphorylation may be necessary but not sufficient for c-fos induction.