An improved method for the purification of high molecular weight urokinase to homogeneity from human urine was established. A yield of 32% with a 3,100-fold purification was obtained by Hyflo Super-Cel treatment, heat treatment at 60 degrees C for 10 hr, serial column chromatography on DEAE-Sepharose CL-6B and O-[3-(p-sulfophenylamino)-2-hydroxypropyl]-cellulose (SFOP-cellulose), and gel filtration on Ultrogel AcA 54. The low molecular weight form of urokinase was also purified to homogeneity by chromatography on hydroxyl apatite and gel filtration on Sephadex G-75 after the SFOP-cellulose column step. The high molecular weight urokinase had only one isoelectric form with a pI of 9.7, whereas the low molecular weight form had six isoelectric subforms with pI values between 9.4 and 6.4. The absorption coefficients at 280 nm of both urokinase forms were 13.61 and 13.50, respectively. Fibrinolytic and esterolytic activities of the two urokinase forms were compared in various assay methods.