Prothrombinase affects the proteolytic conversion of prothrombin to thrombin and is the penultimate enzyme in the common coagulation pathway. Prothrombinase is a complex in which the proteinase, Factor Xa, a cofactor, Factor Va, and calcium are bound to a membrane surface to generate the active enzyme. Guinea pig line 1 and line 10 tumor cells, grown as primary cultures from ascites tumors or as cell lines in culture, provide a surface that interacts with coagulation Factor Va and Xa and with calcium ions to form this enzyme complex. Cultured human colorectal carcinoma cells (Colo 205) also participate in prothrombinase complex assembly and function. Prothrombinase generation was measured by following the kinetics of prothrombin conversion to thrombin. Thrombin generation was monitored continuously using the reversible thrombin inhibitor, dansylarginine N-(3-ethyl-1,5-pentanediyl)amide, which displays enhanced fluorescence upon binding to thrombin. Analyses of kinetic data indicate that the apparent dissociation constants (1-4 X 10(-10) mol/liter) and the number of Factor Va-Xa binding sites per tumor cell are comparable to values reported for human and bovine platelets, human lymphocytes, and monocytes. Guinea pig lymphocytes were also active, while erythrocytes were inactive, in the prothrombinase assay. Membrane vesicles, shed by guinea pig and human tumor cells into conditioned medium, also supported functional prothrombinase activity. Although earlier studies indicated that tumor cells may initiate coagulation, this is the first demonstration that tumor cells are competent to bring clotting to fruition by generating thrombin, a step essential to fibrin generation. These data suggest that tumor cells, in the presence of clotting initiators and appropriate coagulation factors, are sufficient to generate the fibrin deposited in solid tumors.