To evaluate the disposition of fractionated 3H-heparin, high affinity fraction (fraction A) and low affinity fraction (fraction NA) to protamine were prepared in this study from commercial 3H-heparin by affinity chromatography on protamine-Sepharose and gel filtration chromatography. Following intravenous administration of fraction A to rats, it seemed that the radioactivity in plasma declined one-exponentially with time to a pseudo-steady-state level, and 20.5% of dose was excreted in urine in 5 h. However, following the administration of fraction NA to rats, the plasma radioactivity curve was different from that of fraction A, and 69.8% of dose was excreted in urine in 5 h. It was found that fraction A and fraction NA had the distinctly different disposition characteristics in the body. Additionally, whole-body autoradiography with fraction A showed that the liver, bone marrow, kidney cortex, salivary gland and intestine played an important role in the distribution of it. It was suggested by gel filtration chromatography and affinity chromatography that the main material of the radioactivity excreted in urine in 5 h has the same properties as that of the administered heparin fraction.