Single-chain pro-urokinase is an inactive proenzyme form of human urokinase with a single-chain structure and a Mr of 50,000 and converted to the active two-chain form by catalytic amounts of plasmin. It was isolated from culture fluid of human kidney cells and subjected to chemical (CNBr) and proteolytic (lysyl endopeptidase) degradation. The resulting peptides were separated by reverse-phase high performance liquid chromatography and subjected to automated sequence analysis. Amino acid sequence of 85% of the 411 residues recovered in 17 peptides were found to be consistent with those of the A chain (157 amino acids) and B chain (253 amino acids) of human urokinase reported by Günzler and co-workers (Günzler, W. A., Steffens, G.J., Otting, F., Kim, S.-M., A., Frankus, E., and Flohé, L. (1982) Hoppe-Seyler's Z. Physiol. Chem. 363, 133-141; 1155-1165; Steffens, G.J., Günzler, W.A., Otting, F., Frankus, E., and Flohé, L. (1982) Hoppe-Seyler's Z. Physiol. Chem. 363, 1043-1058). It revealed the presence of Lys at position 158 in single-chain pro-urokinase through which the two polypeptide chains of human urokinase are unified into one molecule. In addition, firm evidence was found that upon activation by plasmin single-chain pro-urokinase is cleaved at the Lys-Ile bond between residues 158 and 159, resulting in the formation of a two-chain urokinase molecule held together by one disulfide linkage. These results indicate that the cleavage at the Lys-Ile bond between residues 158 and 159 is responsible for conformational change, appearance of enzyme activity and reduction of its high affinity for fibrin.