A method is described by which the time-course of thrombin generation in plasma can be obtained from a continuous optical density recording of p-nitroaniline (pNA) production in a 2:3 diluted plasma. A chromogenic substrate, methylmalonyl-methylanalyl-arginyl-pNA (SQ 68), is used that is specifically split by thrombin but at a low rate. The thrombin that appears and disappears in the plasma does not split more than 5% of the substrate added, so the rate of substrate conversion is in good approximation proportional to the amidolytic activity in the plasma over the entire period of thrombin generation. The course of the enzyme concentration can be calculated from the amidolytic activity curve. It is shown that the thrombin generation curves obtained in this way are essentially identical to those obtained via the classical subsampling method. The presence of SQ 68 influences the amount of free thrombin that appears in plasma because it competitively inhibits the inactivation of thrombin by AT III and alpha 2 macroglobulin. The inhibition of the thrombin peak by heparin, relative to an uninhibited control, remains unaltered by the presence of the substrate. From the course of thrombin activity and the prevailing decay constants, the course of prothrombin conversion velocity can be calculated. Prothrombin conversion was seen to be inhibited at high (> 500 microM) substrate concentrations only, and experimental conditions are found under which the inhibition of the clotting process by the substrate is negligible. The amidolytic activity is the sum of the activities of free thrombin and of the alpha 2 macroglobulin-thrombin complex formed.(ABSTRACT TRUNCATED AT 250 WORDS)