Since most brain glycoproteins from beta-1,4-galactosyltransferase (beta-1,4-GalT) I knockout mice were galactosylated without apparent reduction the gene expression of novel beta-1,4-GalTs II and V which are involved in N-linked oligosaccharide biosynthesis in addition to beta-1,4-GalT I was studied during mouse brain development. Isolation and characterization of beta-1,4-GalT II and V cDNAs from mouse brains indicates that they are also functioning in the brain. Northern blot analysis revealed that the beta-1,4-GalT I gene is expressed mainly in mid-embryonic stages, while the expression level of beta-1,4-GalT II transcript remains constant and of beta-1,4-GalT V transcript increases during mouse brain development after birth. In situ hybridization revealed that beta-1,4-GalT II and V signals are present in most neural cells, with a marked difference between them in the hippocampus of adult mouse brain tissue. The differential gene expression of beta-1,4-GalTs I, II and V during mouse brain development could affect the differential galactosylation of brain glycoproteins, as revealed by lectin blot analysis.