Abstract A new technique, based on molecular size and charge distribution, was used to assess the heterogeneity of the thrombin standards ( 70 157 and P4) stored at 5 and 37°C as well as ecarin thrombin. Samples were electrophoresed in strips of 1, 2 and 4 % agarose gel which were then cut into 20 equal slices and eluted with buffer before assaying by synthetic substrate (S2238), fibrinogen clotting and platelet rich plasma aggregation methods. To evaluate the influence of contaminant serine proteases assays were also performed using S2222, S2251, S2302 and S2160 in the presence and absence of various inhibitors (AT III, trasylol and hirudin). Standard thrombins were found to be heterogeneous, consisting of both large and small molecular forms with differing amidolytic and biological properties. When degraded at 37°C, only the smaller forms retained some activity. Ecarin thrombin consisted mainly of the larger forms. The observed divergence in the assays for thrombin and the abnormal decay pattern were tentatively attributed to different forms of thrombin and/or prothrombin degradation products, present in the various thrombin preparations.