Objectives The residence time of hyaluronan (HA) in the synovial fluid (SF) of knee joints was investigated using the rabbit anterior cruciate ligament transection (ACLT) model. The aims were to assess at 7 and 28 days after surgery for non-operated (NonOp), ACLT, and SHAM groups: 1) HA molecular mass (Mr) distribution in SF, 2) endogenous replenishment of HA after saline washout, 3) HA residence times in SF, and 4) synovium and subsynovium cellularity. Methods Adult NZW rabbits underwent ACLT or SHAM surgeries on one hind limb, while each contralateral limb was a NonOp control. At 7 or 28d after surgery, joints were aspirated for SF, lavaged with saline, injected with saline or polydisperse HA, and sampled over 8hrs. Fluid samples were analyzed for HA concentration and Mr distribution to calculate HA residence times. Results HA Mr-distributions showed 1) loss of high-Mr HA at day 7, and a shift towards a lower-Mr HA distribution at day 28, 2) endogenous replenishment of high-Mr HA after washout, and 3) Mr-dependent HA loss, particularly at day 7 after ACLT. The residence time of HA decreased with Mr (~27hrs for 7000–2500kDa to ~7hrs for 250–50kDa) and at day 7 after ACLT (~70% decrease). The subsynovium of ACLT joints contained 4) increased cellularity and neovascularization at 7 and 28 days. Conclusions The residence time of HA in SF is transiently decreased after ACLT, suggesting a biophysical transport mechanism for the altered SF composition post-injury or during inflammation.