Abstract: Gangliosides are sialylated glycosphingolipids whose biosynthesis is catalyzed by a series of endoplasmic reticulum (ER)‐ and Golgi‐resident glycosyltransferases. Protein expression, processing, and subcellular localization of the key regulatory enzymes for ganglioside biosynthesis, sialyltransferase II (ST‐II) and N‐acetylgalactosaminyltransferase I (GalNAcT), were analyzed upon transient expression of the two enzymes in the neuroblastoma cell lines NG108‐15 and F‐11. The enzymes were endowed with a C‐terminal epitope tag peptide (FLAG) for immunostaining and immunoaffinity purification using a FLAG‐specific antibody. Mature ST‐II‐FLAG and GalNAcT‐FLAG were expressed as N‐glycoproteins with noncomplex oligosaccharides. ST‐II‐FLAG was distributed to the Golgi apparatus, whereas GalNAcT‐FLAG was found in the ER and Golgi. Inhibition of early N‐glycoprotein processing with castanospermine resulted in a distribution of ST‐II‐FLAG to the ER, whereas that of GalNAcT‐FLAG remained unaltered. In contrast to GalNAcT, the activity of ST‐II and the amount of immunostained enzyme were reduced concomitantly by 75% upon incubation with castanospermine. This was due to a fourfold increased turnover of ST‐II‐FLAG, which was not found with GalNAcT‐FLAG. The ER retention and increased turnover of ST‐II‐FLAG were most likely due to its inability to bind to calnexin upon inhibition of early N‐glycoprotein processing. Calnexin binding was not observed for GalNAcT‐FLAG, indicating a differential effect of N‐glycosylation on the turnover and subcellular localization of the two glycosyltransferases.