In order to estimate the magnitude of electrostatic exclusion provided by the fixed negative charges of the skin and muscle interstitia of rat in-vivo we measured the distribution volumes of two differently charged albumin probes within these tissues. An implanted osmotic pump was used to reach and maintain a steady-state extracellular concentration of a mixture containing two iodine-labeled probes: a charged-modified human serum albumin, cHSA (i.e., a positive probe, pI = 7.6) and a native human serum albumin, HSA (i.e., a normally charged, negative probe, pI = 5.0). Steady-state tissue concentrations were achieved after intravenous infusion of probes for 5 to 7 days. At the end of this period the animals were nephrectomized and a bolus of 51Cr-EDTA was administered for estimating the extracellular volume. Plasma volumes were measured as 5-min distribution volume of 125I - HSA in separate experiments. The steady state interstitial fluid concentrations of all probes were determined using nylon wicks implanted postmortem. Calculations of labeled probes were made for interstitial fluid volumes, (Vi), extravascular albumin distribution volumes, (Va,w), and relative interstitial excluded volume fractions (Vex/Vi). We found that the positive probe is excluded from a significantly smaller fraction of the interstitium. Specifically, the average relative albumin exclusion fractions obtained were: 16% and 26% in skeletal muscle and 30% and 40 % in skin, for cHSA and HSA, respectively. On average, the fixed negative charges of the interstitium are responsible for about 40% of the total albumin exclusion in skeletal muscle and 25% in the whole skin tissue and thus, contribute significantly to volume exclusion in these tissues.