The role of mononuclear cells in generating procoagulant activity was examined by incubating Ficoll-Hypaque-separated mononuclear leukocytes with or without mitogens (phytohemagglutinin, pokeweed mitogen, and concanavalin A). The procoagulant activity was assayed by a modification of a one-stage plasma recalcification time. Significant procoagulant activity developed after 24 hr incubation and was dose dependent; mitogens alone had no effect on the clotting tune. The increase in activity was paralleled by the increase in tritiated thymidine incorporation into replicating DNA. However, mitomycin C had little inhibitory effect on the development of procoagulant activity, whereas thymidine incorporation was inhibited. The major procoagulant activity was associated with intact cells and not the conditioned supernatant. The removal of adherent mononuclear cells (mostly monocytes) by polystyrene bead columns abolished the procoagulant activity, whereas purification of mononuclear leukocyte populations for monocytes markedly increased the activity as compared to purified lymphocytes. The procoagulant activity was shown to act by the extrinsic limb of the coagulation sequence because substitution factor VII-deficient plasma for normal plasma resulted in marked depression of procoagulant activity, whereas factor VIII-deficient plasma resulted in a clotting tune only minimally longer than normal plasma. Thus, although procoagulant activity in cultures of mononuclear cells is stimulated by the mitogen reagent, these studies suggest that the activity may not be the result of the mitogenic effect on lymphocytesper se.Whether it is a direct effect of the mitogen on the adherent cell or is an effect of a contaminant of the mitogen reagent, such as endotoxin, remains to be determined.SpeculationFibrin formation is an important component of inflammatory responses. This study shows that mononuclear leukocytes can be activated to stimulate the formation of fibrin. This suggests that mononuclear leukocytes may play a key role in the deposition of fibrin in inflammatory responses.