Glycosaminoglycan synthesis and secretion by primate arterial smooth muscle have been examined in cell culture. Mass cultures of diploid primate arterial smooth muscle cells were either double labeled with [35S]sulfate and [3H]acetate or single labeled with [3H]glucosamine for 24 h and glycosaminoglycans were extracted and isolated from the culture medium. Incorporation of labeled precursors into glycosaminoglycan was maximal during stationary phase of smooth muscle cell growth in culture and reduced, but not eliminated during logarithmic growth. The glycosaminoglycans synthesized and secreted into the culture medium were characterized by differential susceptibility to glycosaminoglycan-degradative enzymes and by cellulose acetate electrophoresis. Both assay procedures indicate that cultured primate arterial smooth muscle cells synthesize principally dermatan sulfate (60%-80% of total), chondroitin sulfate A and/or C (10%-20%of total) and little or no hyaluronic acid (0%-5% of total). This pattern of glycosaminoglycan formation differed significantly from that exhibited by isologous skin fibroblasts cultured under identical conditions. Dermal fibroblasts synthesize and secrete primarily hyaluronic acid (50%-60% of total) with lesser amounts of dermatan sulfate (10%-20% of total) and chondroitin sulfate A and/or C (10%-20% of total). These results indicate that differences exist in proteoglycan metabolism between these two connective tissue-producing cells in vitro, and suggest that the observed pattern of in vitro glycosaminoglycan synthesis by primate arterial smooth muscle cells may be characteristic for this cell type and not a general response to conditions of cell culture.