The synaptic organization between vesicular glutamate transporter 2 (VGLUT2)-immunoreactive (ir) axon terminals and phrenic motoneurons in the neonatal rat was examined using a combined retrograde tracing and immunohistochemistry for VGLUT2. The phrenic nucleus (PhN) contained large numbers of VGLUT2-ir axon terminals, some of which made axosomatic and axodendritic synapses with PhN motoneurons. These terminals were of asymmetrical type and contained spherical clear synaptic vesicles. The results suggest that in the neonatal rat glutamatergic synapses onto PhN motoneurons exist and mediate excitatory transmission to drive PhN motoneurons.