Hyaluronan (HA) retention inside the synovial cavity of joints serves diverse protective roles. We tested the hypothesis that HA retention is mediated by the network of extracellular matrix proteins in the synovial lining. Cannulated rabbit knee joints were infused with HA solution with/without pre-treatment by chymopapain, a collagen-sparing protease. Trans-synovial fluid escape rate was measured and, after a period of trans-synovial filtration, samples of intra-articular fluid and subsynovial fluid were analysed for HA to assess its trans-synovial ultrafiltration. In control joints HA ultrafiltration was confirmed by post-filtration increases in intra-articular HA concentration (259%+/-17% of infused concentration) and reduced subsynovial concentration (30% +/-8%) (n=11). The proportion of HA molecules reflected by the synovium was 57%-75%. Chymopapain treatment increased the hydraulic permeability of the synovial lining 13 fold; almost abolished the trans-synovial difference in HA concentration; and reduced the HA reflected fraction to 3%-7% (n=6; p<0.001, ANOVA). Structural studies confirmed that chymopapain treatment depleted the matrix of proteoglycans but preserved its collagen. The findings thus demonstrate that HA ultrafiltration and synovial hydraulic permeability are determined by the network of non-collagen, extracellular matrix proteins. This may be important clinically, since protease activity is raised in rheumatoid arthritis, as are HA and fluid escape.