The fibrin specificity of tissue-type plasminogen activator can be increased by mutagenesis within at least four sites in the protease domain. These sites include residue I276, the new N-terminus formed by conversion to a two-chain structure, residues on either side of the active site cleft, KHRR 296-299 or DDD 364-366, a charged surface involved in fibrin interactions, which includes residues H432, R434, D460, R462 and a loop structure, PQANL 466-470, near the fibrin-binding patch. Variants with mutations at any of these sites have low fibrinogen-stimulated activity, whereas fibrin-stimulated activity is at least normal. Kinetic analysis reveals that mutations at these positions reduce the kcat in the presence of fibrinogen, but leave the molecules with normal kinetic constants in the presence of fibrin. A significant exception is found at positions 296-299, where the presence of fibrin manifests significant increases in both kcat and Km. Combinations of mutations at these sites appear to be additive with respect to fibrin specificity.