The mesothelial cell envelopes the surface of the parietal and visceral pleura. These cells are known to synthesize most of the protein constituents of the pleural basement membrane and interstitium. This study examined the ability of a rat pleural mesothelial cell line to synthesize proteoglycans in vitro. Cells were labeled with inorganic 35SO4 to label the glycosaminoglycan moiety of proteoglycans. The medium and combined cell membrane/extracellular matrix fractions contained 73 and 25% of the proteoglycan radioactivity, respectively. The medium contained a single chondroitin/dermatan sulfate proteoglycan of approximately 190 kDa, consistent with biglycan. As determined by Northern analysis of steady-state levels of messenger RNA, the cells contained message for biglycan. Stimulation of the cells with epidermal growth factor resulted in the appearance of a second chondroitin/dermatan sulfate proteoglycan of approximately 97 kDa, characteristic of decorin. The cell membrane/matrix contained a biglycan-like chondroitin/dermatan proteoglycan and several heparan sulfate proteoglycans. Pleural mesothelial cells in vitro are capable of synthesizing a variety of interstitial and basement membrane proteoglycans.