We have developed a solid-phase clotting assay which uses peroxidase-fibrinogen in solution and fibrinogen bound to microtiter plates as a substrate for the thrombin generated from the clotting cascade. We have developed this assay for measurement of the extrinsic pathway factors thromboplastin (tissue factor, factor III), VII and VIIa, X, and II. Using long incubation times (40-90 min), thromboplastin could be measured in extracts of human brain at very low concentrations. Specificity for thromboplastin was demonstrated by showing a requirement for factors II, V, X, and VII but not for VIII, IX, XI, or XII; both substrate plasmas monodeficient in single factors and mixtures of the pure factors were used in demonstrating this specificity. The assay was modified to measure factors II, VII, VIIa, and X using appropriate deficient plasmas. The limit of detection was 2-3 orders of magnitude lower than a one-stage clotting test for all factors assayed. This assay has the advantages of convenience, specificity comparable to standard clotting tests, and high sensitivity.