The synthesis of cartilage link proteins was studied in organ cultures of sterna from normal and nanomelic chick embryos. Nanomelic chondrocytes synthesize cartilage-specific proteoglycans at 1 to 2% of normal levels, and therefore, nanomelic cartilage contains very little proteoglycan aggregate. The defect in proteoglycan synthesis results from a reduced availability of proteoglycan core protein. Link protein synthesis was monitored by the incorporation of [35S]cysteine into protein. Radiolabeled proteins were extracted from cartilage in 4 M guanidine hydrochloride and separated from proteoglycan monomer by centrifugation in dissociative cesium chloride density equilibrium gradients. The top one-sixth (D6) fraction of these gradients contained link protein and was used to extract one unlabeled normal sternum. These extracts were dialyzed to conditions permitting the formation of proteoglycan aggregates and chromatographed on controlled pore glass (CPG 2500). Proteoglycan aggregates chromatograph in the void volume (V0) of these columns. Radioactivity eluting in the CPG 2500 (V0 from normal and nanomelic D6 fractions was identified as link protein by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and subsequent fluorography. Link proteins were also extracted from unlabeled cartilages and identified by the Western blotting technique using link-specific antiserum. Immunoprecipitation of [35S]cysteine-labeled link protein from normal and nanomelic D6 fractions indicated that nanomelic chondrocytes synthesize link protein at normal levels.