Chondrocytes from rabbit ear cartilage were isolated and cultured as monolayers in Ham's F-12 medium. The proteoglycans synthesized by short-term cultures formed a high proportion of aggregates and contained chrondroitin-4- and -6-sulfate in a 2:1 proportion. Dermatan sulfate was not present. The average MW of the condroitin sulfate was about 20,000. Keratan sulfate with an average MW of about 6000 could be isolated from the proteoglycan monomers. Rabbit ear chondrocytes in culture produced proteoglycans comparable to those isolated from hyaline cartilage. Culture for longer periods and plating at lower density caused a decrease in the proportion of aggregated proteoglycans. Primary cultures continued to synthesize aggregated proteoglycans for at least 2 wk; subdivision of the cultures caused a shift toward the production of small-sized ubiquitous proteoglycans. The synthesis of proteoglycan aggregates could be partialy restored by transfer of the monolayer cells to a suspension culture.