Tissue-type plasminogen activator (tPA) mutants which, at selected amino acid positions, mimic urokinase-type plasminogen activator (uPA) were expressed in Chinese hamster ovary cells and examined for their catalytic properties. In one series of mutants, the dipeptide Ser262 Thr263 between kringle 2 and the protease domain of tPA was (a) replaced by an Ala residue, (b) lengthened by additional Ser and Ala residues, (c) exchanged for the 16-amino-acid link between kringle and protease domains of uPA and an additional Ala residue. The activities of the latter two mutants toward plasminogen were, in the absence of fibrin, 3-5-fold higher and, in the presence of fibrin, comparable to or lower than the activity of tPA. The kinetic data suggest a short interdomain peptide in tPA as most favorable for high fibrin stimulation of tPA activity. In a second series of mutant, selected amino acid residues of the tPA protease domain were replaced by residues of the homologous uPA domain. Positions chosen for exchange are either close to the active site or are part of a tPA-specific insertion in the variable region preceding the active-site Ser residue. Compared to authentic tPA, protease-domain mutants exhibited 7.3-424-fold lower activities toward plasminogen, mainly due to lower kcat values. Km values differed only moderately. A mutant containing an additional hydroxyl group at the S1 site, tPA A473S, had lost the preference of tPA for Arg over Lys as the P1 residue in peptide substrates.