Tissue plasminogen activator (tPA) is a single chain glycoprotein of 527 amino acids consisting of structural domains homologous to other plasma proteins ("finger","epidermal growth factor", "kringles" and "protease"). Unlike zymogens of other serine proteases, tPA in the single chain form (1-527), has amidolytic and fibrinolytic activity. However, the amidolytic activity is enhanced when tPA is cleaved by plasmin at the Arg275-Ile276 bond to yield the disulfide bonded two chain form. We used trypsin to study the structure and function of tPA by limited digestion. Aliquots of tPA (1 mg/ml) were digested at pH 7 with varying amounts of trypsin (1:10,000, 1:1000, 1:100 and 1:10; enzyme to substrate ratio). The dilute solutions of trypsin (1:10,000) were effective at completely converting one chain tPA to the two chain form, but little additional proteolysis was observed on SDS-PAGE. The proteolytic fragments of tPA were isolated by reduction and carboxymethylation (RCM), SDS gel electrophoresis and reversed phase HPLC. The RCM polypeptides were identified by amino acid composition and sequence. Specific antisera were prepared against peptide antigens of tPA including (1-27), (1-275), (276-527) and (502525). Immunoblotting experiments with the tryptic digests of tPA indicated that the region (1-275) is more susceptible to proteolytic attack than the protease (275-527). Specific cleavage sites were identified at positions 7, 10, 27 and 40. Partially digested tPA preparations were tested for enzymatic activity as determined by hydrolysis of the peptide substrate S-2288 or by clot lysis. Limited proteolysis at the amino terminus was correlated with significant loss of fibrinolytic . activity but minimal effect on the amidolytic activity. Increased tryptic digestion resulted in complete loss of amidolytic activity and significant reduction in antigenic activity as determined by polyclonal anti-tPA ELISA. These results areconsistent with the amino terminal "finger" domain being in part responsible for the fibrin-binding specificity of tPA. Limited tryptic digest of tPA, cleaves first at Arg-275, then subsequently cleaves the "finger" with associated loss of fibrinolytic activity.