We studied the physiologic roles of the hyaluronidase (HYAL) 1 and HYAL2 in hyaluronan (HA) turnover. HA was localized and quantified using HA binding proteins in various tissues of Hyal1(-/-) and Hyal2(-/-) mice (knockout mice) as well as control mice. HA MW was determined using gel filtration chromatography. HA endocytosis in liver nonparenchymal cells (NPCs) was quantified in vivo. Both Hyal1 and Hyal2 knockout mice showed HA accumulation in peripheral tissues without changes in HA MW distribution. HYAL2 deficiency induced buildup of very high MW (>3.10(6) Da) HA in lymph and serum with severe lymph node distortion. The lack of HYAL2 also impaired high MW HA endocytosis by liver NPCs. HYAL1 deficiency led to a moderate increase in serum HA concentration without changes in HA MW distribution and to HA overload of liver NPCs. Wild-type C57BL/6 mice served as controls. In HA injection experiments, saline-injected mice served as additional controls. We conclude that: 1) HYAL1 and HYAL2 are both needed for tissue HA catabolism; 2) HYAL2 is required for high MW HA clearance in lymph nodes and plasma and for HA endocytosis by liver NPCs; and 3) the main role of HYAL1 is HA degradation within liver NPCs.-Bourguignon, V., Flamion, B. Respective roles of hyaluronidases 1 and 2 in endogenous hyaluronan turnover.