Our previous studies have demonstrated that neurokinin-1 receptor (NK1R)-immunoreactive (ir) neurons in the pre-Bötzinger Complex (pre-BötC), the hypothesized kernel of respiratory rhythmogenesis, receive both glutamatergic excitatory and GABAergic or glycinergic inhibitory inputs. Neuromodulators, such as substance P (SP) and opioids, play important roles in normal respiratory activity and respiratory disorders. The identification of the relationship between neurotransmitters and NK1R-ir neurons at the cellular level is essential for understanding the synaptic interaction within the pre-BötC network. Using immunofluorescence and immunogold-silver staining, we wished to exploit SP and enkephalin (ENK) immunoreactivity and their relationships with glutamate, GABA, glycine, or NK1R in the pre-BötC in adult Sprague-Dawley rats. The pre-BötC contained a substantial amount of SP-ir and ENK-ir boutons. They were largely colocalized with glutamate and much less so with GABA. Glycine immunoreactivity was rarely found in either SP-ir or ENK-ir boutons. A number of SP-ir boutons were ENK-ir as well. Synapses were commonly found between SP-ir or ENK-ir terminals and NK1R-ir neurons in the pre-BötC. Most of them were asymmetric. Symmetric synapses made up 10% of all synapses examined between SP-ir boutons and NK1R-ir neurons, and 19% of ENK/NK1R synapses. Colocalization of SP and/or ENK with glutamate in boutons in the pre-BötC implies the combined synaptic release of excitatory amino acid and neuropeptides, which may exert combined post-synaptic effects onto NK1R-ir neurons and contribute to respiratory activity.