In joint fluid the polymer hyaluronan (HA) confers viscous lubrication and greatly attenuates trans-synovial fluid loss (outflow buffering). Outflow buffering arises from the molecular sieving (reflection) and concentration polarization of HA at the synovial membrane surface. Outflow buffering declines if HA chain length is reduced, as in arthritis, and this has been attributed to reduced HA reflection. This was tested directly in the present study. Infused solutions of HA of approximately 2200 kDa (HA2000, 0.2 mg ml(-1)) or approximately 500 kDa (HA500, 0.2 mg ml(-1)) or approximately 140 kDa (HA140, 0.2-4.0 mg ml(-1)) were filtered across the synovial lining of the knee joint cavity of anaesthetized rabbits at a constant rate, along with a freely permeating reference solute, 20 kDa fluorescein-dextran (FD20). After a priming period the femoral lymph was sampled over 3 h. Mixed intra-articular (i.a.) fluid and subsynovial fluid were sampled at the end. Fluids were analysed by gel exclusion chromatography. The trans-synovial concentration profile was found to depend on polymer size. The i.a. concentration of HA2000 increased substantially relative to infusate and the subsynovial and lymph concentrations fell substantially. For HA500 and HA140 the trans-synovial concentration gradients were less pronounced, and absent for FD. The reflected fractions for HA2000, HA500 and HA140 across the cavity-to-lymph barrier were 0.65 +/- 0.05 (n = 10), 0.43 +/- 0.09 (n = 3) and 0.19 +/- 0.05 (n = 7), respectively, at matched filtration rates (P < 0.0001, analysis of variance). Reflected fractions calculated from HA i.a. accumulation or subsynovial dilution showed the same trend. The results demonstrate size-selective molecular sieving by the synovial extracellular matrix, equivalent to steric exclusion from cylindrical pores of radius 33-59 nm. The findings underpin the concentration polarization-outflow buffering theory and indicate that reduced HA chain length in arthritis exacerbates lubricant loss from a joint.