Whole corneas and the three corneal cell-types (epithelium, endothelium, and fibroblasts) were incubated in vitro for varying lengths of time and then labeled with [ 35S]sulfate. Freshly isolated whole corneas, corneal stromas, and isolated corneal fibroblasts all incorporated [ 35S]sulfate into keratan sulfate-like polysaccharide. Neither the corneal epithelium nor the endothelium incorporated label into this polysaccharide. After 48 hr in vitro under a variety of culture conditions, no tissue or isolated cell population continued to synthesize keratan sulfate-like polysaccharide. The cultured corneal tissues continued to synthesize and secrete chondroitin 4-sulfate as well as heparan sulfate, chondroitin 6-sulfate, and a dermatan sulfate-like material. The latter three polysaccharides are not thought to be normal components of the cornea.