Glycine receptors are chloride-permeable, ligand-gated ion channels and contribute to the inhibition of neuronal firing in the central nervous system or to facilitation of neurotransmitter release if expressed at presynaptic sites. Recent structure-function studies have provided detailed insights into the mechanisms of channel gating, desensitization, and ion permeation. However, most of the work has focused only on comparing a few isoforms, and among studies, different cellular expression systems were used. Here, we performed a series of experiments using recombinantly expressed homomeric and heteromeric glycine receptor channels, including their splice variants, in the same cellular expression system to investigate and compare their electrophysiological properties. Our data show that the current-voltage relationships of homomeric channels formed by the α2 or α3 subunits change upon receptor desensitization from a linear to an inwardly rectifying shape, in contrast to their heteromeric counterparts. The results demonstrate that inward rectification depends on a single amino acid (Ala(254)) at the inner pore mouth of the channels and is closely linked to chloride permeation. We also show that the current-voltage relationships of glycine-evoked currents in primary hippocampal neurons are inwardly rectifying upon desensitization. Thus, the alanine residue Ala(254) determines voltage-dependent rectification upon receptor desensitization and reveals a physio-molecular signature of homomeric glycine receptor channels, which provides unprecedented opportunities for the identification of these channels at the single cell level.