Glycine receptors (GlyRs) play an important role in inhibiting neurone activity in the spinal cord. Until recently adult GlyRs were thought to comprise α1 and β subunits. A new form of the receptor containing α3 subunits has been discovered in the superficial dorsal horn (SDH), a region of the spinal cord important for pain. This raises questions about the precise subunit composition of GlyRs and glycinergic synapses in the SDH. We used the spasmodic mouse, where α1 subunit containing GlyRs have altered agonist sensitivity and electrophysiological properties, to ask how α1 and α3 subunits are assembled to form GlyRs on SDH neurones. We found most (∼75%) GlyRs and glycinergic synapses in the SDH contain α1 subunits and few are composed exclusively of α3 subunits. Therefore, future efforts to design pain drugs that target the α3 subunit must consider the potential interaction between α1 and α3 subunits in the GlyR.