A method for the efficient further purification of two active forms of urokinase (UK) [EC. 3. 4. 21. 31] from partially purified human urinary UK was established by serial column chromatography on Sephadex G-100, Sephadex G-75, and SP-Sephadex C-50. According to Ferguson plots two active forms showed molecular weights of 5.5×104 (H-UK) and 3.6×104 (H-UK). Both UK forms were shown to be homogeneous by SDS-polyacrylamide gel electrophoresis, gel gradient electrophoresis, and Ouchterlony's double immunodiffusion method. Amino acid analysis showed that there was no marked difference in composition between H- and L-UK. The specific activity of H-UK was 1.2×105 IU/mg protein, which exceeds the highest specific activity that has ever been obtained by other investigators, and that of L-UK was 1.52×105 IU/mg protein. These results suggest that this serial column chromatography procedure is effective for the purification of H-UK, which is known to be a pharmacologically advantageous form. As judged by isotachoelectrophoresis, H-UK contained five subforms with pI values of 8.7, 8.9, 9.1, 9.2, and 9.4, and L-UK contained the same number of subforms with pI values of 7.5, 8.3, 8.8, 9.4 and 9.7. The pI values of the subforms with the highest specific activity were 9.4 for H-UK and 8.3 and 8.8 for L-UK. Judging from the dissimilarity in the pI values of UK preparations obtained so far by other investigators and ourselves, we consider that the subforms of UK obtained after purification do not correspond to the forms present in vivo, but are artifacts of the purification procedures used.