The aim of this study was to examine the inhibitory effect of high molecular weight hyaluronan (HA) on nuclear factor (NF)-kappaB activation by COOH-terminal heparin-binding fibronectin fragment (HBFN-f) in rheumatoid arthritis (RA) chondrocytes. When RA chondrocytes in monolayer or cartilage explants were cultured with HBFN-f, the fragment stimulated the phosphorylation and nuclear translocation of NF-kappaB, leading to nitric oxide (NO) production in association with inducible form of NO synthase (iNOS) up-regulation. Inhibition studies with NF-kappaB inhibitors indicated the requirement of NF-kappaB for HBFN-f-induced NO production. Pretreatment with 2700 kDa HA resulted in significant suppression of NF-kappaB activation by HBFN-f. HA also inhibited HBFN-f-stimulated NO production with down-regulation of iNOS. The present study clearly demonstrated that high molecular weight HA suppressed HBFN-f-activated NF-kappaB in RA chondrocytes. HA could down-regulate the catabolic action of fibronectin fragments like HBFN-f in RA joints as a potent NF-kappaB inhibitor.