The tetra-alanine substitution variant KHRR 296-299 AAAA of tissue-type plasminogen activator (t-PA) was previously shown to have enhanced fibrin specificity and enhanced activity in the presence of fibrin compared with the wild-type form of the molecule. The structural requirements for these alterations in enzymatic activity were investigated by constructing several amino acid substitution variants at each of the positions from 296 to 299 and evaluating their activities under a variety of conditions. Effects on plasminogen activator activity were common among the point mutants at positions 296-299; nearly all had a phenotype similar to the KHRR 296-299 AAAA variant. The greatest effects on enzymatic function were found with multiple substitution variants, but some single charge reversals and proline substitutions had substantial effects. The enhanced fibrin specificity of KHRR 296-299 AAAA t-PA results in less fibrinogenolysis than seen with wild-type t-PA. Approximately four times greater concentration of KHRR 296-299 AAAA compared with wild-type t-PA was required to consume 50% of the fibrinogen in human plasma.