Brain-derived neurotrophic factor (BDNF) is involved in the modulation of synaptic transmission in the spinal cord, and several circumstantial lines of evidence suggest that it has the ability to modulate the activity of the NMDA receptor. Here we dissect the signalling mechanisms by which BDNF exerts its neuromodulatory role on the NMDA receptor subunit 1 (NR1). Using a preparation of adult isolated dorsal horn with dorsal roots attached, we found that electrical stimulation of roots induced a concomitant release of BDNF and an increased phosphorylation of NR1, which was partly prevented by the BDNF sequestering molecule, TrkB-IgG. Using a second approach in vitro, we confirmed that both exogenous glutamate and BDNF (but not other neurotrophins) were able to induce NR1 phosphorylation, in particular at residue Ser-897. NR1 phosphorylation induced by BDNF was blocked by a TrkB inhibitor, an ERK inhibitor and a PKC inhibitor but not a PKA inhibitor. Activation of PKC using exogenous PMA also led to NR1 phosphorylation. Together these data suggest that BDNF modulates the activity of the receptor by phosphorylation via the kinases ERK and PKC.