The radiochemical purity of 131I-hippuran determined by radiochromatographic analysis using Whatman No 3 MM paper and a mixture of n-butanol, acetic acid and water as developer, was 97.3 +/- 1.2%. O-131IB acid and less than 1% of free 131I were separated from the preparation. No radioactivity which would correspond to O-131IB acid was observed on the radiochromatogram when 131I-hippuran was incubated with HSA and heparin. Protein binding of 131I-hippuran as a function of radiochemical purity, and HSA and heparin concentrations were determined by the method of equilibrium dialysis. No effect of radiochemical purity on protein binding of 131I-hippuran was observed, the value obtained being 1.3 +/- 0.1%. Higher HSA concentrations increased protein binding but decreased retention activity on the dialysing membrane. Heparin decreased protein binding of 131I-hippuran by 47%. The biological half-times of excretion of 131I-hippuran with heparin were 4 min for the fast phase and 107 min for the slow phase. The results of biodistribution in experimental animals showed increased localization of 131I-hippuran with heparin in the kidneys and liver during the first 15 min after injection.