Adenosine 2a (A2a) receptor agonists elicit persistent increases in phrenic nerve activity by transactivating the neurotrophin receptor, TrkB, near phrenic motoneurons. Our working model proposes that A2a receptor-mediated TrkB receptor activation strengthens glutamatergic synapses onto phrenic motoneurons. Activation of glutamate NMDA receptors has been implicated in other models of phrenic motor plasticity. Thus, we hypothesized that NMDA receptor activation also would contribute to A2a receptor-mediated phrenic motor facilitation. Adult male Sprague Dawley rats were anesthetized with urethane, mechanically ventilated, neuromuscularly paralyzed, and bilaterally vagotomized. The A2a receptor agonist, CGS-21680, and the NMDA receptor-channel blocker, MK-801, were administered intrathecally over the C4 spinal segment. Phrenic nerve activity was recorded before, during, and after drug administration. MK-801 (concentration range 0.1, 1.0, 10.0, and 100 µM) was administered 30 min before CGS-21680 (50 µM). MK-801 dose-dependently blocked A2a receptor-mediated phrenic motor facilitation. When administered at 60 min post-CGS-21680, MK-801 prevented further increases in phrenic nerve activity compared to the CGS-21680 alone (CGS-21680 alone at 120 min: 114 ± 19 %, CGS-21680 and MK-801 at 60 min post-CGS-21680: 61 ± 11 %, above baseline, p<0.05), but did not return phrenic motor output to baseline values. Our data suggest that NMDA receptor activation is necessary for de novo A2a receptor-mediated phrenic motor facilitation and that the maintenance of pre-existing phrenic motor facilitation does not involve NMDA receptor-dependent mechanisms.