A novel proteoglycan (PG) has been identified in culture medium from thin slices of the superficial zone of bovine articular cartilage. This PG is synthesized and secreted selectively by chondrocytes of this zone but has not been demonstrated in culture medium from slices deeper in the same tissue. There is little, if any, incorporation of this PG into the extracellular matrix. The PG has been partially purified by isopycnic CsCl density gradient ultracentrifugation, ion-exchange chromatography on DEAE Sephacel, and gel filtration chromatography on Sepharose CL-2B. It migrates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of approximately 345 kDa. The molecule is degraded by papain, trypsin, or pronase; however, limited pepsin treatment performed at 4 degrees C only decreases its molecular weight to approximately 315 kDa. The molecule is substituted with keratan sulfate and chondroitin sulfate, which are largely removed by limited pepsin treatment. In addition, this PG, or a very similar molecule, has been demonstrated in synovial fluid. This novel PG may serve as a functional metabolic marker for chondrocytes of the superficial zone of articular cartilage.