Hyaluronan is a major component of the vitreous gel. Hyaluronan-binding macromolecules, including the aggregating proteoglycans, have been shown to perform an important role in maintaining the structural integrity of a number of tissues. However, there have not previously been any biochemical data to establish the presence of these types of macromolecules in vitreous. Bovine vitreous gel was solubilized (apart from a residual collagenous pellet) in 4 M guanidine hydrochloride and after dialysis into phosphate buffered saline analyzed by gel filtration chromatography with in-line measurement of refractive index and multi-angle laser light scattering. The concentration of hyaluronan in whole vitreous was found to be 0.57 mg/ml. The average molecular weight of the hyaluronan was found to be 170,000 (after isolation of the vitreous hyaluronan by isopycnic centrifugation in 0.5 M guanidine hydrochloride and papain digestion). Following Superose 12 gel filtration chromatography of the Streptomyces hyaluronan lyase digested vitreous extract, a pool of material was identified at or near the void volume of the column, and this material was shown to contain sulphated proteoglycans. Analysis of fractions following Superose 12 gel filtration chromatography by Western blotting showed that this pool of material contained the chondroitin sulphate proteoglycans versican and type IX collagen. Link protein was also identified in vitreous extracts by Western blotting. In whole vitreous, the concentration of versican was found to be 21.4 ± 2.8 μg/ml and of link protein 0.62 ± 0.07 μg/ml. Versican and link protein were thus present in approximately 1:1 molar ratios, but hyaluronan was present in a molar excess of 150 times. Therefore, aggregating proteoglycans are present in vitreous but, assuming that they bind to hyaluronan in-vivo, their overall density along the hyaluronan is much lower than that found in other tissues.