Mechanical compression of cartilage and tendon has been shown to expel fluid both from collagen fibrils and from the extrafibrillar space. As reported previously, albumin (Alb) concentration and colloid osmotic pressure in tendon fluid (TF) expelled by repeated centrifugations fell progressively at increasing centrifugation force (G = 600, 2,400, and 13,100), suggesting either molecular sieving in compressed tendon or mobilization of protein-free (excluded) fluid. The present experiments, including analysis of 51Cr-EDTA, aprotinin (Ap), Alb, immunoglobulin G (IgG), and hyaluronan (hyaluronic acid; HA) with molecular weight (MW) ranging from 341 to 5 x 10(6), strongly favored the exclusion hypothesis; the fraction of Alb, IgG, and HA-free fluid (excluded) was already 0.23-0.36 in the first centrifugate, increasing to 0.73-0.82 in the third. The corresponding numbers were, respectively, 0.11 and 0.43 for Ap (MW 6,500), and 0 and 0.08 for 51Cr-EDTA. These data, combined with calculated exclusion by collagen fibrils, proteoglycans, and HA, indicated that the first centrifugate was mainly derived from the extrafibrillar space, with increasing addition of macromolecular free intrafibrillar fluid in the second and third centrifugates, with each space contributing about equally to the total centrifugate volume. The calculations also indicated that Alb-, IgG-, and Ap-free fluid was mobilized from extrafibrillar space by increasing overlap of excluded territories. An excess of HA in tendon compared with that estimated from centrifugate concentrations suggests a large bound or immobilized HA fraction.