We recently reported on a brain-specific beta1,6-N-acetylglucosaminyltransferase IX (GnT-IX, also referred to as GnT-VB), a GnT-V homologue, which acts on alpha-linked mannose of N-glycans and O-mannosyl glycans. To distinguish functions of GnT-IX with GnT-V, we examined the distribution of GnT-IX and GnT-V transcripts in mouse tissues by Northern blot analysis. The two enzymes were differentially expressed as has previously been observed in human tissues. GnT-IX transcripts were restricted to the cerebrum, cerebellum, thymus and testis, whereas GnT-V transcripts were expressed ubiquitously in mouse tissues. To investigate the localization of these enzymes in mouse tissues in more detail, a polyclonal antibody against GnT-IX was prepared. The antibody specifically recognized GnT-IX, but not GnT-V, in the Golgi apparatus, as confirmed by the use of GnT-IX and GnT-V transfectants. In agreement with the Northern blot analysis data, an immunohistochemical study showed substantial expression of GnT-IX in the brain, while no expression was observed in the liver. Moreover, to exclude GnT-V contamination, we performed an enzymatic assay for GnT-IX using a Mgat5 (GnT-V)-null mouse brain as an enzyme source and found the enzymatic activities do, in fact, exist in mouse brain. The reaction product was confirmed by high performance liquid chromatography and mass spectrometry. These results demonstrate, for the first time, that GnT-IX protein is actually expressed and may function as a glycosyltransferase in the brain.