A new solid-phase thrombin (EC 3.4.21.5) was prepared through conjugation of the enzyme under mild conditions to a glass support bearing an active ester of N-hydroxysuccinimide. The immobilized enzyme retained 50 +/- 10% of the specific esterase activity of the parent soluble enzyme. The Km (apparent) for the esterase activity of the immobilized enzyme has a value of 5 mM, identical of the Km value of the parent-soluble enzyme. Only 6 +/- 1% of the specific proteolytic activity was retained and a higher Km (apparent) value of 67 muM was obtained for the insoluble enzyme compared to Km value of 12.5 muM for the parent soluble thrombin. Solid-phase thrombin prepared by the diazocoupling technique was previously reported to retain only 3% of the specific proteolytic activity. The observed loss of specific proteolytic activity can be attributed to steric interference, a change in charge characteristics, or both. Nevertheless, the present method of preparation has the advantages of rapidity and simplicity. It can readily be adapted to use for studying the fate of various complexes of fibrinogen, fibrin and their degradation products. It should also be useful for preparing radiolabeled autologous soluble fibrin for thrombus detection in patients undergoing active thrombosis.