The ultraviolet absorption difference spectrum of heavy meromyosin induced by ATP was followed in the presence of 10−3 M MnCl2. The life time of the difference spectrum was almost one second regardless of the initial concentration of ATP. This time was far longer than that of the initial burst of Pi-liberation of ATPase [EC 3.6.1.3]. The difference spectrum after decay was in the shape of an ADP-induced one. On addition of MgCl2 (final concentration, 10−2 M) to heavy meromyosin solution containing 10−4M MnCl2 at a time when an adequate concentration of ATP still remained, the shape of the difference spectrum, which was already in the form of an ADP-induced one, changed immediately to that of an ATP-induced one. The binding of ADP to heavy meromyosin was measured by the gel-filtration method in the presence of MnCl2. The Scatchard plot was concave upwards and the maximum binding number obtained was 2.1 moles per 3.65 xl05g of heavy meromyosin. Comparison between the degree of binding and magnitude of the difference spectrum induced by ADP showed that the maximum value of A A was attained at one mole of ADP binding. The maximum value of the initial burst of Pi-liberation was about 1.3 moles per 3.65×l05g of heavy meromyosin in the presence of MnCl2. The initial burst decreased through the addition of ADP, without any change in the steady state ATPase rate. Based on these results, the mechanism of ATPase action is discussed.