Cultures of embryonic fibroblasts from Balb/c or CBA/J mice were given 12-h pulses of 14C-galactose, or were double-labelled with 3H-galactose and 35H-sulfate. The time course of the rates of labelling of glycosaminoglycans--galactose label was found in the uronic acid moiety--was studied in synchronously and asynchronously growing cultures. Partial synchrony was achieved by trypsinising quiescent, confluent cells and subsequent transfer of cells to new cultures with fresh medium. Synchrony was monitored by measurement of thymidine uptake in parallel cultures. The distribution of label in the hyaluronic acid, chondroitin sulfate, and heparan sulfate fractions from cells and culture media was determined at each time point. Peaks of DNA synthesis were accompanied by or followed 12 h later by a maximal rate of labelling with galactose of secreted glycosaminoglycans, and with the exception of hyaluronic acid--also of cellular glycosaminoglycans. The rate of labelling with galactose of glycosphingolipids in parallel cultures followed a different time course. In double-label experiments the rates of labelling of glycosaminoglycan sulfates with 3H-galactose and 35S-sulfate did not go parallel. In older, quiescent cultures the labelling rate with galactose decreased while the sulfation rate increased. It is discussed that the labelling rate with galactose is indicative of the biosynthetic rate of the glycosaminoglycans. The conclusion is reached that glycosaminoglycans are preferentially synthesized and secreted after the S phase of the cell cycle.