The proteoglycan components of Bruch's membrane have not been well characterized to date. In this study, the glycosaminoglycans present in Bruch's membrane were identified and found to be heparan sulfate with small amounts of chondroitin and/or dermatan sulfate and hyaluronic acid. The biosynthesis of glycosaminoglycans and their incorporation into proteoglycans was investigated using an eye organ culture in which the cornea, iris, and sclera had been removed. The newly synthesized proteoglycan(s) extracted with 4 M guanidine hydrochloride bound to a DEAE-cellulose column and eluted with approximately 0.44 M NaCl. The proteoglycan(s) had a molecular weight ranging from 100,000 to 150,000 daltons. After papain treatment, the glycosaminoglycan side chains had a molecular weight of approximately 44,000 daltons. The newly synthesized proteoglycan(s) contained 65% chondroitin and/or dermatan sulfate and 35% heparan sulfate. This organ culture system should be useful in studying disease states of Bruch's membrane.