Inhibition of thrombin proteolysis of fibrinogen with D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone (PPACK) results in irreversible inactivation of the thrombin catalytic site, but the PPACK-inhibited thrombin, through its exosite, retains its ability to bind to fibrinogen or fibrin. Hirudin inactivates thrombin at the catalytic site and also inhibits thrombin exosite binding to fibrin or fibrinogen. PPACK or hirudin was added to a clotting mixture of fibrinogen and active thrombin (enzyme:substrate ratio, 1:400 and 1:800) prior to the onset of gelation. Subsequent fibrin assembly was evaluated by turbidity measurements at 350 nm and by determining the fibrin and fibrinogen content of the clots that ultimately formed. Polymerization rates and the fibrin/fibrinogen content of the clots that formed were greater in the PPACK-inhibited system than in the hirudin-inhibited system. Lowering the ionic strength from 0.14 to 0.09 amplified these differences. The results suggest that in addition to its well-recognized role in the proteolytic conversion of fibrinogen to fibrin, thrombin functions as a cofactor in the fibrin assembly process.