Proteoglycans and hyaluronic acid synthesized by human skin fibroblasts in culture were characterized, and the effect of thyroid hormone deficiency was examined. The fibroblasts in culture synthesize hyaluronic acid, dermatan sulfate (DS) proteoglycans and heparan sulfate (HS) proteoglycans. Hyaluronic acid is almost exclusively secreted into the medium. Among the proteoglycans synthesized during 24 h label, about 70% were secreted into the medium and the remaining 30% were associated with the cell layer. About 70% of proteoglycans secreted into the medium contained DS and the remaining 30% contained HS. For cell-associated proteoglycans, 60% contained HS and the remainder contained DS. The size distributions of the glycosaminoglycans from both DS and HS proteoglycans were similar, with an average Mr of approximately 30,000. Incubation of fibroblasts in thyroid hormone deficient medium increased net synthesis of hyaluronic acid (approximately 50%) and all species of proteoglycans (approximately 85%). 3H/35S ratios in the chondroitin 4-sulfate disaccharide isolated with HPLC were not altered in thyroid hormone deficient cultures, indicating that the specific activity of 3H in UDP-N-acetylhexosamine precursors did not change. The increased incorporation of 3H into hyaluronic acid and of 3H and 35S into DS and HS proteoglycans thus indicates increased net synthesis. Degradation of cell-associated proteoglycans was not influenced by thyroid hormone deficiency.