The distribution of the three glycosyltransferases synthesizing the terminal trisaccharide sialic acid yields D-galactose yields N-acetylglucosamine present in many glycoproteins was determined in Golgi fractions prepared from rat liver homogenates by a modification of the procedure of Ehrenreich et al. (1973, J. Cell Biol. 70:671--684). The enzymes were assayed with asialofetuin, ovomucoid, and Smith-degraded ovomucoid as sugar acceptors. Careful adjustment of the pH of all sucrose solutions to 7.0 +/- 0.1 prevented enzyme inactivation, and allowed quantitative recoveries at every isolation step. The three morphologically and functionally different Golgi fractions GJ1, GF2, and GF3 showed (in that order) decreasing specific activities of all three enzymes, but the relative amounts and relative specific activities of the three transferases in any given fraction were nearly identical. Two marginal fractions, one extra heavy (collected on the gradient below GF3) and the other extra light (isolated by flotation from the postmicrosomal supernate) were found to contain recognizable Golgi elements. An enrichment of any transferase over the two others was not detected in either preparation. A partial release of content from a combined GF1+2 was achieved by treatment with the nonionic detergent Triton X-100. Low Triton/phospholipid ratios (less than 2 mg/mg) led to lysis of the vesicles and cisternae and loss of very low density lipoprotein particles (ascertained by electron microscopy), but failed to separate the transferases from each other; the three enzymes sedimented together with a population of empty vesicles to a density of approximately 1.08 g/ml.