A technique is presented for the preparation of three major proteoglycans from 14-day embryonic chicken retinas following their culture overnight with [35S]sulfate and either [3H]glucosamine or [3H]serine. Homogenization of the tissue in saline permitted extraction of heterogeneous soluble proteoglycans separately from most of the heparan sulfate proteoglycans. The latter were extracted from the 140,000g pellet with 0.5% Triton X-100 in 8 M urea. The medium plus the saline and urea-detergent extracts were separated from low-molecular-weight contaminants, and fractionated into two peaks of radioactivity on Sephacryl S-300 in saline with 3 M urea and 0.5% Triton X-100. The proteoglycans were isolated directly from these fractions on DEAE-Sephacel, and subjected to ultrafiltration concentration and then further purification on cesium chloride density gradient centrifugation in 4 M guanidine hydrochloride. A further step involving cetylpyridinium chloride precipitation was examined, but it resulted in essentially no further purification. The fractionations separated a large chondroitin sulfate/dermatan sulfate proteoglycan from the culture medium that was excluded from S-300 and of low buoyant density; a large heparan sulfate proteoglycan from the urea-detergent extract that was also excluded from S-300 and of low buoyant density; and two smaller and possibly related heparan sulfate proteoglycans. One was found in the medium and showed low to intermediate buoyant density; the other was isolated from the urea-detergent extract and showed a significantly higher buoyant density, associated with a lower protein content. The saline extract contained both of the two larger proteoglycans and only minor amounts of the smaller molecules.