High- and low-molecular-weight forms of urokinase have been prepared by affinity chromatography. The amino-terminal sequence of 47 residues of the latter has been determined and its homology compared with other serine proteases. The primary structure using classical procedures is being determined. Fluorescence studies of both forms of UK with dansyl fluoride and dansyl peptide chloroketone indicate that they have identical catalytic sites and are also more apolar than trypsin with a slight difference at another binding site.