Mouse mastocytoma cells grown in suspension culture produce chondroitin 4-sulphate. A Golgi-apparatus-enriched fraction from these cells was prepared and examined for chondroitin-synthesizing activity. When Golgi-apparatus-enriched fractions were incubated with UDP-[14C]glucuronic acid and UDP-N-acetylgalactosamine, they demonstrated a greater than 13-fold increase in chondroitin-synthesizing activity over cell homogenates. Similar incubations with the addition of a pentasaccharide from chondroitin sulphate resulted in a greater than 40-fold increase in [14C]glucuronic acid-incorporating activity over cell homogenates. Other membrane fractions had much less activity, suggesting that the Golgi apparatus is the most active location for chondroitin biosynthesis. Products of the incubations indicated the formation of [14C]chondroitin glycosaminoglycan on endogenous primers and formation of [14C]-hexasaccharide and somewhat larger [14C]oligosaccharides on exogenous pentasaccharide acceptors. There was, however, a significant amount of large [14C]-chondroitin glycosaminoglycan formed on pentasaccharide, indicating that some pentasaccharide did serve as a true primer for polysaccharide synthesis.