Our previous study demonstrated GABAergic and glycinergic synapses onto neurokinin-1 receptor (NK1R)-immunoreactive (ir) neurons in the pre-Bötzinger complex (pre-BötC), the hypothesized kernel of normal respiratory rhythmogenesis. In the present study, we aimed to identify glutamatergic synapses onto NK1R-ir pre-BötC neurons, as excitatory synaptic transmission is a prerequisite to normal respiratory rhythmogenesis. Two types of vesicular glutamate transporters (VGLUT), VGLUT1 and VGLUT2, have been recently implicated in glutamate-mediated transmission. The present study used immunofluorescence and immunogold-silver staining to determine the relationship between the transporters and NK1R-ir neurons in the pre-BötC of adult rats. Under the confocal laser-scanning microscope, VGLUT2-ir boutons were found to be widely distributed in the pre-BötC, some of which were in close apposition to NK1R-ir somas and dendrites. VGLUT1-ir boutons were relatively rare and only a few were found to be in close apposition to NK1R-ir somas and dendrites. Electron microscopic observation revealed that approximately 41% of VGLUT2-ir terminals were in close apposition to, or made asymmetric synapses with NK1R-ir somas and dendrites in the pre-BötC. On the other hand, 50.5% of NK1R-ir dendrites were closely apposed to, or synapsed with VGLUT2-ir terminals. Occasionally, VGLUT1-ir terminals were found in close apposition to NK1R-ir somas or dendrites, but we were unable to identify synapses between them. The present findings provide the morphological basis for excitatory synaptic inputs onto NK1R-ir neurons in the pre-BötC. VGLUT2 may be involved in a dominant excitatory synaptic pathway for normal respiratory rhythmogenesis.