Sepharose CL-4B chromatography of guanidine hydrochloride and aqueous extracts of 3H-glucosamine labeled intact corneal tissue reveals four peaks representing proteoglycans and glycoproteins. To evaluate the universality of the 4th peak, hereafter designated as Sepharose CL-4B (IV), its presence was investigated in rabbit, bovine, cat, rhesus monkey, and human corneal preparations. Following incubation in isotopically labeled medium, corneas were extracted with aqueous and/or 4M guanidine hydrochloride and subjected to Sepharose CL-4B chromatography. Sepharose CL-4B (IV) was detected in all species studied; 3H-glucosamine and 14C-amino acids, but not 35SO4, were incorporated into this peak which eluted in the range consistent with an apparent molecular weight of approximately 30,000 D. To determine which layers were involved in the synthesis of Sepharose CL-4B (IV) the layers of the rabbit cornea were incubated separately (stroma scraped of endothelium and/or epithelium, epithelium only, endothelium only). A distinct Sepharose CL-4B (IV) peak was not identified in the chromatographs obtained from organ cultures of corneal epithelium, endothelium, or from corneal stroma scraped of epithelium and/or endothelium. This decrease in Sepharose CL-4B (IV) synthesis occurred even if the scraped cornea was not allowed to expand in volume by compressing it beneath a membrane porous to the incubation medium. Thus, Sepharose CL-4B (IV) synthesis was enhanced significantly by the stroma being in conjunction with other corneal cells as they exist in vivo.