ldlD cells, which lack the UDP-Gal/UDP-GalNAc 4-epimerase, were stably transfected with a Myc-tagged version of N-acetylglucosaminyltransferase I (Myc-Glc-NAc-T I). In the absence of GalNAc and Gal, newly synthesized GlcNAc-T I did not acquire O-linked oligosaccharides but was catalytically active and was transported to the Golgi region as defined using both immunofluorescence and immunoelectron microscopy. After addition of cycloheximide to prevent further synthesis, GalNAc and Gal were added, and the unglycosylated GlcNAc-T I was found to acquire mature, O-linked oligosaccharides with a half-time of about 150 min. The addition of these sugars was sensitive to N-ethylmaleimide and okadaic acid, both inhibitors of vesicle-mediated traffic. Together, these results suggest that Myc-Glc-NAc-T I undergoes retrograde transport to the early part of the Golgi apparatus where the first O-linked sugar, GalNAc, is added followed by anterograde transport back to the Golgi stack, where addition of Gal and sialic acid occurs.