The extracellular matrix of normal liver contains several types of proteoglycans including heparan sulphate, chondroitin sulphate isomers, dermatan sulphate, and the glycosaminoglycan, hyaluronic acid. In the present study both the synthesis and secretion as well as the pattern of radioactively labeled proteoglycans and hyaluronic acid of hepatocytes, fat-storing cells (Ito cells), and Kupffer cells maintained in monolayer cultures under mostly identical conditions were compared to assess their relative contribution to hepatic proteoglycan synthesis. Fat-storing cells were identified as the main type of cell producing and secreting proteoglycan and hyaluronic acid. More than 70% of labeled proteoglycan and hyaluronic acid were secreted into the medium. Heparan sulphate is the main type of proteoglycan in hepatocytes, whereas in the medium of fat-storing cells, chondroitin sulphate and dermatan sulphate comprise the major fractions. Hyaluronic acid was not detectable in hepatocyte cultures and found only in low amounts in the medium of Kupffer cells. The results point to a stringent quantitative and qualitative cellular compartmentation of proteoglycan synthesis in liver with fat-storing cells as the most important cell type for matrix proteoglycan and hyaluronic acid production.