Persistent hyperalgesia is associated with increased expression of proteins that contribute to enhanced excitability of spinal neurons, however, little is known about how expression of these proteins is regulated. We tested the hypothesis that Substance P stimulation of neurokinin receptors on spinal neurons activates the transcription factor nuclear factor of activated T cells isoform 4 (NFATc4). The occurrence of NFATc4 in spinal cord was demonstrated with RT-PCR and immunocytochemistry. Substance P activated NFAT-dependent gene transcription in primary cultures of neonatal rat spinal cord transiently transfected with a luciferase DNA reporter construct. The effect of Substance P was mediated by neuronal neurokinin-1 receptors that coupled to activation of protein kinase C, l-type voltage-dependent calcium channels, and calcineurin. Interestingly, Substance P had no effect on cyclic AMP response element (CRE)-dependent gene expression. Conversely, calcitonin gene-related peptide, which activated CRE-dependent gene expression, did not activate NFAT signaling. These data provide evidence that peptides released from primary afferent neurons regulate discrete patterns of gene expression in spinal neurons. Because the release of Substance P and calcitonin gene-related peptide from primary afferent neurons is increased following peripheral injury, these peptides may differentially regulate the expression of proteins that underlie persistent hyperalgesia.