BackgroundThe glycinergic system plays an important inhibitory role in the mouse central nervous system, where glycine controls the excitability of spinal itch- and pain-mediating neurons. Impairments of the glycine receptors can cause motor and sensory deficits. Glycine exerts inhibition through interaction with ligand-gated ion channels composed of alpha and beta subunits. We have investigated the mRNA expression of the glycine receptor alpha 3 (Glra3) subunit in the nervous system as well as in several peripheral organs of female and male mice.ResultsSingle-cell RNA sequencing (scRNA-seq) data analysis on the Zeisel et al. (2018) dataset indicated widespread but low expression ofGlra3in vesicular glutamate transporter 2 (Vglut2, Slc17a6) positive and vesicular inhibitory amino acid transporter (Viaat, Slc32a1)positive neurons of the mouse central nervous system. Highest occurrence ofGlra3expression was identified in the cortex, amygdala, and striatal regions, as well as in the hypothalamus, brainstem and spinal cord. Bulk quantitative real-time-PCR (qRT-PCR) analysis demonstratedGlra3expression in cortex, amygdala, striatum, hypothalamus, thalamus, pituitary gland, hippocampus, cerebellum, brainstem, and spinal cord. Additionally, male mice expressed higher levels ofGlra3in all investigated brain areas compared with female mice. Lastly, RNAscope spatially validatedGlra3expression in the areas indicated by the single-cell and bulk analyses. Moreover, RNAscope analysis confirmed co-localization ofGlra3withSlc17a6orSlc32a1in the central nervous system areas suggested from the single-cell data.ConclusionsGlra3expression is low but widespread in the mouse central nervous system. Clear sex-dependent differences have been identified, indicating higher levels ofGlra3in several telencephalic and diencephalic areas, as well as in cerebellum and brainstem, in male mice compared with female mice.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12868-023-00800-9.