The glycine receptor (GlyR) is an inhibitory neurotransmitter-gated ion channel found mainly in the brainstem and spinal cord. Alternative splicing results in 2 isoforms of the GlyR α3 subunit denoted as short (S) and long (L), which differ by 15 amino acids in the second intracellular loop (residues 358-372). The GlyR α3L has a significantly decreased potency for glycine and a significantly reduced rate and extent of desensitisation that was no longer voltage-sensitive. The underlying molecular basis for these differences was investigated using whole-cell patch-clamp recording from wild-type and mutant rat GlyR α3 subunits expressed in human embryonic kidney (HEK) cells. Residues 365-372 were important in reducing the potency of glycine as their substitution or deletion resulted in a 2-3-fold increase in glycine potency. These residues were also important determinants of the slower desensitisation rate of the GlyR α3L, their deletion increasing the rate of desensitisation 3-fold. The presence of the charged residues lysine (K) 365, arginine (R) 368 or aspartate (D) 371 with the remaining residues between 365-372 substituted by alanines produced GlyRs with a 2-4-fold increase in the potency of glycine compared to that at the GlyR α3L. Therefore, the remaining residues between 365-372 are involved in producing the lower potency of glycine at the GlyR α3L. The presence of all three charged residues restored slow desensitisation to the GlyR α3L. A combination of charged residues are important determinants for the differing desensitisation properties of the GlyR α3L but are not responsible for lowering the potency of glycine. The large intracellular loop is implicated in the pharmacological and kinetic properties of the GlyR α3. The increased specialisation of the GlyR α3 may be of functional importance for glycinergic neurotransmission in various regions of the central nervous system including auditory, retinal and spinal cord neurones.