A simple assay method for platelet factor 4 is described. When factor Xa was added to a system containing antithrombin III in excess and heparin in low concentration, the amount of factor Xa immediately inactivated was found to be a function of the concentration of heparin. When an antiheparin such as platelet factor 4 was added, an increase of the residual activity of factor Xa was observed. The magnitude of this increase was shown to be correlated to the amount of heparin inactivation in the system. Platelet factor 4 could be assayed when the concentrations of antithrombin III, heparin, and factor Xa were maintained at a constant level and in excess. As an indicator of the reaction, factor Xa was measured with the chromogenic substrate benzoyl-Ile-Glu-Gly-Arg-p-nitro-anilide (S-2222).