The role of phospholipids in the activity of UDP-D-galactose: D-xylose galactosyltransferase (galactosyltransferase I) from embryonic chick cartilage was investigated. Phospholipase C treatment of particulate galactosyltransferase I caused inactivation of this enzyme to the extent of 60 to 70% as well as hydrolysis of 75 to 80% of the membrane phospholipids. Addition of phospholipid restored activity to nearly control levels. The order of effectiveness of various phopholipids in reactivating phospholipase C-treated galactosyltransferase I was as follows: lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylethanolamine, phosphatidylcholine. The effect of phospholipase A on galactosyltransferase I activity was also examined and was found to be concentration-dependent. At concentrations less than 10 mug/mg of pellet protein, phospholipase A slightly activated galactosyltransferase I. whereas at higher concentrations it inhibited the activity in a manner similar to phospholipase C. Galactosyltransferase I was activated moderately and also solubilized by treatment with Nonidet P-40 in the presence of 0.5 M KCl. Following solubilization and purification by gel filtration and affinity chromatography, galactosyltransferase I could be inactivated by detergent removal by dialysis and subsequently reactivated by addition of detergent. Neither phospholipase C treatment nor exogenous phospholipid had any significant effect on three of the other chondroitin sulfate glycosyltransferases (UDP-D-xylose: core protein xylosyltransferase, UDP-D-glucuronic acid:3-O-beta-D-galactosyl-D-galactose glucuronosyltransferase, and UDP-N-acetyl-D-galactosamine:(BlcUA-GalNAc-4-sulfate)j N-acetylgalactosaminyltransferase). On lipid analysis by thin layer chromatography, phosphatidylcholine and phosphatidylethanolamine were found to be the major phospholipids of particulate and solubilized glycosyltransferase preparations from embryonic chick cartilage, while lysopholipids of particulate and solubilized glycosyltransferase preparations from embryonic chick cartilage, while lysophosphatidylcholine and lysophosphatidylethanolamine were barely detectable components. The concentration of these specific phospholipids was diminished greatly following phospholipase C treatment.