Previously, it was demonstrated that the embryonic corneal epithelium produces the chondroitin sulfate and heparan-sulfate-like compounds and the collagen of the primary corneal stroma. Synthesis of all of these extracellular materials is greatly enhanced in vitro when isolated epithelium is grown on collagenous substrata instead of Millipore filters. We report here that chondroitin sulfate, heparin, and heparan sulfate added to the culture medium at a concentration of 200 mug/ml enhance the synthesis by the epithelium of chondroitin sulfate and heparan-sulfate-like compounds 2-fold, whether or not collagenous substrata are employed. Collagen synthesis is unaffected by adding glycosaminoglycan to the medium. Chondroitin sulfate proteoglycan (chondromucoprotein) has the same stimulatory effect as chondroitin sulfate, but dermatan sulfate and hyaluronate have no measurable effect on glycosaminoglycan production by epithelial cells. Keratan sulfate however, seems to depress glycosaminoglycan synthesis. Thus, in this system, only sulfated polyanions like those produced by the corneal epithelium have a stimulatory effect on glycosaminoglycan synthesis. The results are discussed in terms of how the tissues of the cornea (epithelium, endothelium, keratocytes) may interact by changing the composition of the stromal extracellular matrix.