We have studied some aspects of the regulation of hyaluronic acid synthesis in cultured human skin fibroblasts derived from normal individuals. In these cells 85% of radiolabeled glucosamine is incorporated into two types of macromolecules, hyaluronic acid and glycoproteins, characterized by district partitions between the cell and medium compartments. Treatment of cells with cycloheximide under conditions in which protein and glycoprotein synthesis is inhibited by at least 90% has no effect on hyaluronic acid synthesis. This result is consistent with the hypothesis that protein synthesis is not necessary for the synthesis of hyaluronic acid. Conversely, 5 X 10(-6) M dexamethasone phosphate inhibits hyaluronic acid by 60% but has no effect on protein or glycoprotein or glycoprotein synthesis. Concomitant treatment with dexamethasone phosphate and cycloheximide restores the synthesis of hyaluronic acid to normal values. The dexamethasone phosphate effect on hyaluronic acid synthesis is therefore not a primary induction response since it requires protein synthesis. The secretion of hyaluronic acid and glycoproteins from the cells into the medium is not affected by either of the two drugs. This experimental approach will be useful in the study of the regulation of glycosaminoglycan metabolism in human cells especially with respect to disorders of connective tissue.