Corneal explants with scleral rims were freshly prepared from 3-day-old hatched chicks, cultured in vitro for 5 h in the presence of [35S]sulfate and [3H]glucosamine, then the constituent tissues (stroma, epithelium, endothelium, and corneo-scleral rim) were dissected from the explants. Incorporation of 35S activity into stromal tissue was the highest among the constituent tissues (54% of the total). Two predominant proteoglycans were identified in the stromal fraction: keratan sulfate proteoglycan and chondroitin sulfate/dermatan sulfate proteoglycan. Whereas heparan sulfate proteoglycan was found in epithelial tissue as the major proteoglycan therein, it was a minor component in the other tissues. In contrast, most of the 35S-labeled material in the culture medium was the sulfated glycoprotein, which was probably synthesized and secreted by epithelial cells. When each constituent tissue was separately cultured in the presence of radioactive precursors, incorporation of radioactivities into each tissue decreased markedly compared with those into the respective tissues obtained by dissection after culture of the whole cornea with scleral rim. This suggests that corneal constituent tissues interact with each other for proteoglycan synthesis. But the glycosaminoglycan compositions of stromal proteoglycans synthesized in stroma cultured alone and in stroma from the cultured cornea with scleral rim were similar to each other; the two major glycosaminoglycans were chondroitin sulfate/dermatan sulfate and keratan sulfate. In contrast, when stromal tissue was cultured alone, most of the 35S-labeled material in the culture medium was keratan sulfate proteoglycan and free keratan sulfate chain, suggesting that keratan sulfate species are catabolized and eliminated more readily from collagen lamina than chondroitin sulfate/dermatan sulfate species, unless the corneal stroma is surrounded with other tissues. Whereas keratan sulfate was not detected in epithelium cultured alone, a small amount of it was found in endothelium cultured alone.