The synthesis of proteoglycans by primary cultures of 19-day gestation fetal rat lung Type II pneumonocytes was studied. The cells were grown in the presence of [3H]-glucosamine and/or [35S]-Na2SO4 and the radioactive label incorporated into proteoglycans was analyzed. Proteoglycans of high molecular weight (approximately 200 Kd) were isolated by gel permeation chromatography and contained both [3H] and [35S]. The glycosaminoglycan composition of the proteoglycans was determined by electrophoresis and autoradiography. The medium contained 65-80% of the labeled proteoglycans and was enriched for hyaluronate, with lesser amounts of the sulfated glycosaminoglycans (dermatan sulfate greater than heparan sulfate greater than chondroitin sulfate). The cell layers retained 20-35% of the labeled proteoglycans and was enriched for heparan sulfate, with lesser amounts of chondroitan sulfate greater than dermatan sulfate greater than hyaluronate. The synthesis of proteoglycans was time-dependent and was stimulated by increasing concentrations of fetal bovine serum. Cortisol inhibited proteoglycan synthesis, apparently by decreasing the availability of proteoglycan core-protein.