Spinal trigeminal nucleus (Vsp) consists of three subnuclei: oralis (Vo), interpolaris (Vi) and caudalis (Vc). Previous anatomical studies using antero-/retro-grade tracers have suggested that intersubnuclear ascending/descending synaptic transmissions exist between subnuclei. However, pharmacological properties of the intersubnuclear synaptic transmission have not been studied yet. Since three subnuclei are located in Vsp along rostro-caudal axis, it will be necessary to prepare horizontal brainstem slices to perform pharmacological analysis of the intersubnuclear synaptic transmission. We here show horizontal brainstem slices retaining three subnuclei, and that, using blind whole-cell recordings in the slices, synaptic transmission may be abundantly retained between subnuclei in the horizontal slices, except for the transmission from Vo to Vc. Finally, pharmacological analysis shows that excitatory and inhibitory synaptic responses, respectively, are mediated by AMPA and NMDA receptors and by GABA(A) and glycine receptors, with a differential contribution to the synaptic responses between subnuclei. We therefore conclude that horizontal brainstem slices will be a useful preparation for studies on intersubnuclear synaptic transmission, modulation and plasticity between subnuclei, as well as, further, other brainstem nuclei.