Vitreous fibrosis was induced in rabbit eyes by intravitreal injection of monocytes and lymphocytes. The fibrotic vitreous and normal vitreous removed from experimental animals were then incubated with [3H]-glucosamine at 37 degrees C for 24 hr. The newly synthesized 3H-labeled glycosaminoglycans (GAGs) were isolated by 4 M GuHCl extraction followed by pronase digestion. The 3H-labeled GAGs were then characterized by gel-filtration column chromatography and by specific enzymatic degradation, i.e. hyaluronidase, chondroitinase AC and/or chondroitinase ABC. The disaccahrides derived from chondroitinase ABC degradation were identified by thin-layer chromatography. Our results indicated that 91% of the total glycosaminoglycan synthesized by normal vitreous was hyaluronic acid. In contrast, in the fibrotic vitreous, the synthesis of hyaluronic acid was decreased to 30% whereas the synthesis of chondroitin sulfate increased to 47% of the total newly synthesized glycosaminoglycans. Control vitreous which was injected with freeze-thawed monocytes and lymphocytes synthesized 70% hyaluronic acid and 12% chondroitin sulfate although no fibrosis was observed.