The inhibitory glycine receptor is a pentameric membrane protein composed of α and β subunits. In the postsynaptic membrane, the glycine receptor and the copurifying peripheral membrane protein gephyrin are clustered underneath glycine-releasing nerve terminals. Here, we describe the expression of gephyrin and the neonatal and adult glycine receptor α subunit isoforms α1 and α2 during in vitro differentiation of rat spinal neurons. Analysis by immunoassays and the reverse transcriptase—polymerase chain reaction showed that gephyrin and α subunit mRNA and protein levels exhibited a marked increase from 1 to 5 days in vitro, i.e. prior to the formation of functional synaptic contacts. Using confocal and standard immunofluorescence, we determined the number of immunoreactive cells and the cellular localization of the α subunits and gephyrin. At 3 days in vitro, glycine receptor immunoreactivity revealed by the monoclonal antibody mAb4a was found in >10% of cells and was mainly localized intracellularly; in contrast, gephyrin was detected in <50% of cells. At 7 days in vitro, gephyrin was essentially localized at the neuronal surface. At this stage, the number of glycine receptor-positive cells approached that of gephyrin-containing neurons (50%), and glycine receptor antigen was found both intracellularly and at the periphery of the cells. The antibody mAb2b, which binds exclusively to the α1 subunit, revealed aggregates at the surface of a few neurons. At 10 days in vitro, glycine receptor and gephyrin staining was localized in clusters at the periphery of the soma and the neurites. This quantitative analysis corroborates temporal differences in the cellular distribution of gephyrin and glycine receptor α subunits, the former being accumulated first at the neuronal surface.