Following incubation of sealed, "right side out," rat liver Golgi-derived vesicles with a mixture of [3H]GDP-fucose and GDP-[14C]fucose, the difference in the 3H to 14C ratio between the incubation medium and the lumen of the vesicles was less than 11%, suggesting that the sugar nucleotide was transported intact into the vesicles. Transport of GDP-fucose weas temperature-dependent, saturable, with an apparent Km of 7.5 microM and a Vmax of 14 pmol/mg of protein/10 min and inhibited by substrate analogues. Pretreatment of intact Golgi vesicles with pronase inhibited transport by 80% under conditions in which sialyltransferases (a lumenal marker) were not inhibited. This result is consistent with a sugar nucleotide carrier protein, portions of which face the cytoplasmic side of Golgi vesicles. Previous studies from this laboratory had shown that CMP-N-acetylneuraminic acid (CMP-NeuAc) could penetrate mouse liver microsomes in a manner suggesting carrier-mediated transport (Carey, D. J., Sommers, L. W., and Hirschberg, C. B. (1980) Cell 19, 597-605). Subsequent to transport, a portion of the N-acetylneuraminic acid moiety became covalently linked to proteins facing the lumen of Golgi vesicles (Carey, D. J., and Hirschberg, C. B. (1981) J. Biol. Chem. 256, 989-993). Upon subfractionation of rat liver into vesicles derived from the smooth and rough endoplasmic reticulum and Golgi, both the highest CMP-NeuAc specific transport activity and total transport activity were localized in the Golgi. Any transport activity in the other vesicles could be accounted for by contamination with Golgi, as determined by established marker enzyme activities. Transport of CMP-NeuAc was temperature-dependent, inhibited by pronase, substrate analogues, and saturable, with an apparent Km of 2.4 microM and a Vmax of 150 pmol/mg of protein/10 min.