The size of hyaluronan was compared between tissue and lymph using a combination of agarose gel electrophoresis and a radiometric assay. Prenodal lymph was collected from heel skin and the gastrocnemius muscle in anesthetized rabbits. The major fraction of hyaluronan in both tissues had a molecular weight greater than 4 million. Lymph contained primarily low molecular weight hyaluronan (less than 0.79 x 10⁶) which was absent from tissue. Volume loading produced a preferential increase in the flux of low molecular weight hyaluronan, indicating that tissue contains a small quantity of mobile, low molecular weight hyaluronan. The maximum daily removal of hyaluronan by lymph was less than 1% of the tissue content. The amount of lysosomal hyaluronidase activity in tissue was more than enough to account for a rapid turnover of hyaluronan. The data support the conclusion that lymph drainage is not significant in the normal catabolism of hyaluronan and may represent a small amount that becomes detached from the pericellular and extracellular matrices.