We have reported that three different Fn fragments (Fn-f) added to bovine articular cartilage cultured in serum-free DMEM cause marked elevation of proteoglycan (PG) degradation and release into the culture media. We report here that the PG release required the continual presence of Fn-f, that PG release still occurred when serum-free cultures were switched to bovine synovial fluid media, and that addition of recombinant IGF-1, TGF-beta, and recombinant interferon gamma to cultures did not affect Fn-f-mediated PG release. The Fn-f caused a 25-fold enhanced release of stromelysin-1 protein from cartilage by Day 1 and up to 120-fold by Day 3. The stromelysin form released was 43 kDa, the activated form of pro-stromelysin-1. This stromelysin form apparently played a major role in Fn-f-mediated PG release, since addition of Sepharose-bound anti-stromelysin-1 to cartilage cultures greatly slowed rates of PG release. Potential activators of pro-stromelysin-1, plasmin, and u-PA (urinary plasminogen activator), were also detected in conditioned media of Fn-f-treated cartilage. u-PA levels were increased in the presence of the Fn-f but by only a few fold. Addition of alpha-1-antiproteinase inhibitor, which can block enzymatic activity of u-PA, was found to inhibit about half the PG-releasing activity of the Fn-f. Levels of TIMP-1, the 30-kDa tissue inhibitor of metalloproteinases, which can inhibit stromelysin, doubled within 24 h when a Fn-f was added to culture. These data suggest that stromelysin-1 may be a major mediator of Fn-f-mediated PG release from cartilage.