We have estimated exclusion of albumin from the interstitial matrix in rats based on measurement of albumin concentration in interstitial fluid, interstitial fluid volume (IFV), and interstitial albumin mass. Measurements were performed separately in dermis and subcutis during control, overhydration (saline infusion corresponding to 10% of body weight), and dehydration. Exclusion during control was 1.03 (SD = 0.47; n = 7 rats) and 0.85 (SD = 0.21; n = 7 rats) ml/g fat-free dry wt in subcutis and dermis, respectively. This corresponded to 44.1 and 57.8% of IFV, respectively. Collagen and hyaluronan were responsible for two-thirds and one-third of the exclusion, respectively. During overhydration the increase in albumin available volume was 70% of the increase in IFV. Both the collagen and the hyaluronan excluded compartment of the excluded volume changed when altering IFV. These data suggest that the colloid osmotic pressure in the albumin available compartment can change the volume of both the collagen and hyaluronan compartments of the excluded volume. The following changes in composition of the interstitial matrix were observed: albumin mass in subcutis decreased by 20% during dehydration and increased by 30% during overhydration, and overhydration reduced hyaluronan content by 20% in subcutis.