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Methods have been developed for kinetic studies of the inhibition of exogenous unmodified thrombin in human plasma containing normal levels of fibrinogen and calcium ion. To prevent interference by other proteases, factor VIII-deficient plasma was used and contained 50 nM Phe-Phe-Arg-chloromethyl ketone and 1 kallikrein-inactivating unit/ml aprotinin; neither inhibited thrombin at these levels. Two independent assays were used. The first was the discontinuous amidolytic assay of thrombin activity, which measures both free thrombin and thrombin-alpha 2-macroglobulin complex, and was used to estimate the rates of inhibition both by "inactivating" inhibitors, such as anti-thrombin and alpha 1-protease inhibitor, and by alpha 2-macroglobulin (alpha 2M). The contribution of alpha 2M was confirmed by a second method, which measured with time the generation of amidolytic activity due to the thrombin-alpha 2M complex. The total rate of thrombin inhibition in plasma containing 4 mM free Ca2+ was of the order of 1.9 min-1, of which 0.4 min-1 was due to alpha 2M and 0.9 min-1 was due to inhibitors that were removed when plasma was passed through heparin-agarose. Thrombin inhibition was also measured in varying dilutions of plasma and confirmed that total inhibition rate is approximately linearly related to plasma (and thus inhibitor) concentration. Negatively charged phospholipid micelles had very little effect on thrombin inhibition rate, but platelets accelerated inhibition to more than 2.5 min-1.