Previous studies have shown that neurons in the sacral dorsal commissural nucleus (SDCN) express neurokinin-1 receptor (NK1R) and can be modulated by the co-release of GABA and glycine (Gly) from single presynaptic terminal. These results raise the possibility that GABA/Gly-cocontaining terminals might make synaptic contacts with NK1R-expressing neurons in the SDCN. In order to provide morphological evidence for this hypothesis, the triple-immunohistochemical studies were performed in the SDCN. Triple-immunofluorescence histochemical study showed that some axon terminals in close association with NK1R-immunopositive (NK1R-ip) neurons in the SDCN were immunopositive for both glutamic acid decarboxylase (GAD) and glycine transporter 2 (GlyT2). In electron microscopic dual- and triple-immunohistochemistry for GAD/GlyT2, GAD/NK1R, GlyT2/NK1R, or GAD/GlyT2/NK1R also revealed dually labeled (GAD/GlyT2-ip) synaptic terminals upon SDCN neurons, as well as GAD- and/or GlyT2-ip axon terminals in synaptic contact with NK1R-ip SDCN neurons. These results suggested that some synaptic terminals upon NK1R-expressing SDCN neurons co-released both GABA and Gly.