The antifibrinolytic activity of cytosols obtained from cultured rabbit endothelial cells was studied to determine whether it resulted from the presence of an antiplasmin or an antiactivator. These cytosol preparations inhibited the fibrinolytic activity initiated by some plasminogen activators (e.g. urokinase, rabbit endothelial cell activator), but not others (e.g. activators associated with bovine endothelial cells and Rous sarcoma virus-infected chick embryo fibroblasts), suggesting that inhibition occurred at the level of plasmin formation, not plasmin activity. The fibrinolytic activity of plasmin itself was unaffected by concentrations of cytosol that completely blocked urokinase-mediated fibrinolysis consistent with this conclusion. In addition, the ability of urokinase to cleave 125I-plasminogen into its characteristic activation fragments was inhibited by cytosol in a dose-dependent manner. When urokinase was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, two peaks of activity were detected, corresponding to Mr = 55,000 and 32,000. Urokinase preincubated with cytosol and analyzed in a similar manner demonstrated no activity in any portion of the gel, suggesting that its ability to function as a plasminogen activator was irreversibly lost following its interaction with cytosol. These results indicate that the antifibrinolytic activity of rabbit endothelial cells results from the presence of a molecule or molecules with antiactivator activity. The cellular location and unusual degree of specificity distinguish the endothelial cell inhibitor(s) from antifibrinolytic agents observed in other cells and in plasma and platelets.