A low concentration of hyaluronan (HA) in lymph compared with tissue suggests a large bound fraction. To investigate the distribution and mobility of HA and serum albumin (Alb), we eluted the rat tail tendon with a series of l5 successive centrifugations, each preceded by the addition of 0.15 M NaCl (15% of initial wet wt). The eluate concentration fell exponentially versus the accumulated eluate, allowing estimation of the maximal elutable amount (E_HA and E_Alb). Alb elution was practically complete from a space of 28% of wet wt at all centrifugation rates. Twenty percent of HA was elutable at 500 rpm, apparently from the same space as Alb, increasing to 40% at >4,000 rpm. This pattern was not significantly influenced by using 2 M NaCl or by the addition of plasma or metabolic inhibitors. Without prehydration and centrifugation at high revolutions per minute, both Alb and HA concentrations fell rapidly toward zero, presumably in part reflecting mobilization of HA- and Alb-free fluid from the collagen intrafibrillar space (3). We conclude that with prehydration the fibrils swell, increasing the intramolecular spaces to become "penetrable" to HA and allowing removal of HA-containing fluid when the fibrils are compressed by the next centrifugation at high revolutions per minute, increasing E_HA from 23 to 45%. Chemical binding presumably explains the unelutable 55% of tendon HA. Intrafibrillar HA may act to stabilize the fibrillar volume.