Uptake and metabolism of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) by rough-endoplasmic-reticulum (rER)-derived vesicles was studied. Analysis of the molecular species, double-label experiments, cis-inhibition and trans-stimulation experiments revealed that uptake represented entry of intact UDP-GlcNAc into the microsomal lumen. The amount of vesicle-associated label at equilibrium uptake was directly proportional to the volume of the intravesicular space, and permeabilized microsomes were unable to retain UDP-GlcNAc. These findings indicate that uptake constituted effective translocation from the medium into the lumen of the vesicles. The microsomal uptake of UDP-GlcNAc met the criteria of bidirectional, carrier-mediated translocation. Transport was time and temperature dependent, saturable, selective, capable of trans-stimulation and operational against a concentration gradient. Uptake studies performed in membrane preparations that were highly enriched in either rER, smooth ER, or Golgi revealed that UDP-GlcNAc was taken up by the ER and by the Golgi apparatus.