F(1)-ATPase is a rotary molecular motor that makes 120 degrees stepping rotations, with each step being driven by a single-ATP hydrolysis. In this study, a new reaction intermediate of F(1)-ATPase was discovered at a temperature below 4 degrees C, which makes a pause at the same angle in its rotation as when ATP binds. The rate constant of the intermediate reaction was strongly dependent on temperature with a Q(10) factor of 19, implying that the intermediate reaction accompanies a large conformational change. Kinetic analyses showed that the intermediate state does not correspond to ATP binding or hydrolysis. The addition of ADP to the reaction mixture did not alter the angular position of the intermediate state, but specifically lengthened the time constant of this state. Conversely, the addition of inorganic phosphate caused a pause at an angle of +80 degrees from that of the intermediate state. These observations strongly suggest that the newly found reaction intermediate is an ADP-releasing step.