The glycosaminoglycans hyaluronan (HA), heparin, and chondroitin sulfate were biotinylated using biotin-x-hydrazide (biotin-epsilon-aminocaproyl hydrozyde) in conjunction with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride, an activating agent for carboxyl groups. The biotin-x-hydrazide was shown to be coupled directly to the glycosaminoglycans in enzymatic digestions and competition experiments. The biotinylated HA was shown to bind to link protein and receptor for hyaluronic acid-mediated motility, two proteins known to bind HA. The labeled HA was used as a probe to detect known HA-binding proteins in chicken cartilage extract and to identify new HA-binding motifs in the G3 domain of the proteoglycan aggrecan. The significance of the biotinylation of HA, heparin, and chondroitin sulfate A are discussed.