We recently showed that murine peritoneal macrophages cultured in vitro express potent prothrombinase activity (Lindahl, U., Pejler, G., Bøgwald, J., and Seljelid, R. (1989) Arch. Biochem. Biophys. 273, 180-188). In the present report, we demonstrate that the macrophages also express anticoagulant activity by inactivating the thrombin that is formed due to the action of the prothrombinase. Addition of exogenous purified thrombin to the macrophage cultures resulted in inactivation of the enzyme at a maximum rate of approximately 5 micrograms/h/10(6) cells. The inactivation appeared to be specific for thrombin, since neither Factor Xa, chymotrypsin, nor trypsin, three serine proteases exhibiting homology with thrombin, were inactivated by the macrophages. Thrombin-inactivating activity was not secreted into the culture medium. Inhibitors of endocytosis did not decrease the rate by which thrombin was inactivated, suggesting that internalization of the coagulation factor was not required. In contrast, the thrombin-inactivating activity was strongly inhibited by the polycation Polybrene. Anion-exchange chromatography of extracts obtained after Triton X-100-solubilization of the macrophages demonstrated that the thrombin-inactivating activity exhibited a high negative charge. Incubation of the thrombin-inactivating activity recovered after anion-exchange chromatography with unlabeled thrombin, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, showed that thrombin was proteolytically cleaved into defined fragments. Similar proteolytic fragments were obtained when 125I-labeled thrombin was added to macrophage cultures. Degradation of thrombin was blocked by phenylmethanesulfonic fluoride, an inhibitor of serine proteases, but not by inhibitors of other classes of proteases. Thrombin that had been chemically modified at its active site was degraded at the same rate by the macrophages as active thrombin. Taken together, these findings indicate that the murine macrophages express surface-bound serine protease activity that specifically inactivates thrombin by proteolytic cleavage. The significance of thrombin-inactivating activity in relation to the involvement of macrophage procoagulant activity in the immune response is discussed.