Summary Objective To identify the major aggrecanase- and matrix metalloproteinase (MMP)-generated aggrecan fragments in human osteoarthritis (OA) synovial fluid and in human OA joint cartilage. Method Aggrecan fragments were prepared by CsCl gradient centrifugation. Fragment distributions were compared with aggrecanase-1 (ADAMTS-4) and MMP-3 digested human aggrecan by analysis with neoepitope antibodies and an anti-G1 domain antibody, using Western immuno-blots. Results The overall fragment pattern of OA synovial fluid aggrecan was similar to the fragment pattern of cartilage aggrecan cleaved in vitro by ADAMTS-4. However, multiple glycosaminoglycan (GAG) containing aggrecanase and MMP-generated aggrecan fragments were identified in OA synovial fluid and some of these fragments were produced by the action of both types of proteinases. The synovial fluid content of large size aggrecan fragments with 374ARGS- and 342FFGV- N-terminals was about 107 and 40 pmoles per ml, respectively, out of a total concentration of aggrecan fragments of about 185 pmoles per ml. OA synovial fluid contained insignificant amounts of the G1-IPEN 341 fragment as compared to the G1-TEGE 373 fragment, while OA cartilage contained significant amounts of both fragments. OA cartilage contained several GAG-containing aggrecan fragments with N-terminals of G1- or 342FFGV- but no fragments with an N-terminal of 374ARGS-. Conclusions The overall pattern of aggrecan fragments in human OA synovial fluid and cartilage supports an important role for aggrecanase in aggrecan degradation. However, the fragment patterns and their differential distribution between cartilage and synovial fluid are consistent with the existence of at least two proteolytic pathways for aggrecan degradation in human OA, generating both 342FFGV- and 374ARGS-fragments.