Meizothrombin is a transient intermediate produced during the activation of prothrombin by the prothrombinase complex. Because meizothrombin is very sensitive to further activation and autolysis, its isolation is possible only in the presence of active site thrombin inhibitors. This complicates studies of the activities and functions of meizothrombin. As a model, we have expressed a mutant human prothrombin cDNA (R155A, R271A, R284A) with three of the cleavage sites modified so that they are no longer cleaved by factor Xa or thrombin. Several stable baby hamster kidney cell lines were isolated that secreted up to 20 micrograms/ml of carboxylated mutant prothrombin. After purification, the mutant prothrombin was activated by the prothrombinase complex or by ecarin, resulting in the formation of a meizothrombin-like molecule. Electrophoretic analysis and NH2-terminal sequence analysis were consistent with cleavage of a single bond between Arg320-Ile321 and proper processing of the prepropeptide. The meizothrombin was stable for weeks at 4 degrees C. Activation in the presence of dansylarginine N-(3-ethyl-1,5-pentanediyl) amide confirmed the conversion of prothrombin via meizothrombin. Compared with human plasma-derived thrombin, recombinant meizothrombin demonstrated approximately 7% clotting activity, 100% p-toluene-sulfonylarginine methyl ester esterase activity, and approximately 35% S2238 amidolytic activity, and could attenuate fibrinolysis.