Abstract The antithrombin III/thrombin reaction was enhanced by the monovalent cations lithium, sodium and potassium. In the presence of 0.1 M cation, the maximum relative rate enhancement by heparin was 2000-fold with lithium, 1,800-fold with sodium and 1,100-fold with potassium. In the absence of additional cation, heparin enhanced the antithrombin III/thrombin reaction rate 100-fold. The amount of heparin required to maximally enhance the antithrombin III/thrombin reaction rate increased as the concentration of cation was increased. A 10-fold higher concentration of heparin was required in the presence of 0.2 M lithium chloride, while a 100-fold and 150-fold higher concentration of heparin was necessary with 0.2 M sodium chloride and 0.2 M potassium chloride present, respectively. The binding of heparin to antithrombin III was not detectably affected by 0.13 M lithium chloride, suggesting that the heparin-antithrombin III interaction was not involved in the mechanism of action of heparin in this system. The results demonstrate the sensitivity of the heparin-antithrombin III/thrombin reaction rate to small increases in cation concentration and suggest that heparin activity assays using this system must be standardized to accomodate the contribution of cations present in the heparin samples being assayed.