Urokinase inhibitor was purified 120folds from human placental extract by acid treatment, ammonium sulfate precipitation, removal of water insoluble precipitate, adsorption of impurities on CM-Sephadex C-50, gel filtration and hydroxylapatite column chromatography. At the final step, the inhibitor was separated into two fractions (I and II) which were eluted with 0.1M and 0.2 M phosphate buffer (pH6.8) respectively. Trypsin inhibition, enhancement of streptokinase activity, and thromboplastic activity, which were demonstrated in the crude placental extract, were not observed in the purified fraction. Inhibition against urokinase-activated plasmin remained to a negligible degree. It was also proved that the urokinase inhibitor from placenta was different from plasmin inhibitors in human plasma. Urokinase inhibitor showed the electrophoretic behavior as α-globulin on cellulose acetate membrane. Being easily soluble in water, the inhibitor was considered to be pseudoglobulin. By isoelectric focusing, both inhibitor fractions I and II showed one similar isoelectric point (around pH4.8–4.9). The inhibitor having another isoelectric point (pH5.8) was present in a sample before purification on hydroxylapatite column. The inhibitor in each fraction was resolved in two sub-fractions by gel filtration for molecular size analysis. The molecular weight of inhibitor was estimated to be about 70, 000 in the first sub-fraction and about 43, 000 in the second (main) sub-fraction. Urokinase inhibitor solution was stable at 40°C or a lower temperature in a pH range of 5.0–7.5, and also completely stable at 2°C for 12 months.