About 55% of the acyl groups of dog and human vitreous are unsaturated fatty acids. The major components are oleate (18: 1, n-9) and arachidonate (20: 4, n-6) with moderate amounts of linoleate (18: 2, n-6) and docosahexaenoate (22: 6, n-3). Palmitate (16: 0) and stearate (18: 0) are the major saturated fatty acids. There are no significant changes between ages 37–82 years in the fatty acyl group content and composition of human vitreous. In vitreous from Irish setters with hereditary rod-cone dysplasia (RCD) the levels of oleate are decreased with a concomitant increase in arachidonate. [1-−14C] Arachidonic acid was actively incorporated into canine vitreous glycerolipids both in vitro and in vivo. The incorporation was mainly into phosphatidylinositol, triacylglycerol, phosphatidylcholine and phosphatidylethanolamine. There were some differences in the pattern of incorporation between human and dog and between in vivo and in vitro incubations of canine vitreous. Glycerolipid acylation was significantly increased in phosphatidylinositol and phosphatidylcholine in RCD canine vitreous. The pattern of incorporation of [U-14C]docosahexaenoic acid into vitreous glycerolipids was different from arachidonic acid incorporation. Although vitreous did not produce any measurable enzymatic synthesis of cyclooxygenase and lipoxygenase products from [1-−14C]-arachidonic acid in vitro, there was significant generation of autooxidation products. These results suggest an active lipid metabolism in vitreous.