Transmembrane protein 50b, Tmem50b, previously referred to as C21orf4, encodes a predicted transmembrane protein and is one of few genes significantly over-expressed during cerebellar development in a Down syndrome mouse model, Ts1Cje. In order to assess potential mechanisms by which Tmem50b could contribute to Down syndrome-related phenotypes, we determined the expression patterns of Tmem50b mRNA, as well as Tmem50b protein distribution, expression and subcellular localization. In situ hybridization in mice at embryonic day 14.5 showed cortical plate and spinal cord mRNA expression. By postnatal day 7, strong mRNA expression was seen in the cerebellum, hippocampus and olfactory bulb, with diffuse cortical expression. Quantitative PCR of adult mouse tissue showed Tmem50b mRNA expression in the brain, heart and testis. A rabbit polyclonal antibody was generated against Tmem50b and rat and mouse tissue screening by Western blot, and immunohistochemistry showed that protein expression concurred with mRNA expression. Double immunofluorescence revealed that Tmem50b is highly expressed in rat and mouse glial fibrillary acidic protein-positive cells in vivo and in vitro, but less so in neuronal MAP2- or beta-tubulin II-positive cells in vitro. Tmem50b is invariably expressed in cultured mouse neural precursor cells. In adult mouse cerebellum sections, Tmem50b immunoreactivity was found in Purkinje and Golgi cell somata and in Bergmann glial processes. Electron microscopy confirmed that Tmem50b was present on endoplasmic reticulum (ER) and Golgi apparatus membranes. Results indicate that Tmem50b is a developmentally-regulated intracellular ER and Golgi apparatus membrane protein that may prove important for correct brain development through functions associated with precursor cells and glia.