The F₁-ATPase, the soluble part of FoF₁-ATP synthase, is a rotary molecular motor consisting of α₃β₃γδε. The γ and ε subunits rotate relative to the α₃β₃δ sub-complex on ATP hydrolysis by the β subunit. The ε subunit is known as an endogenous inhibitor of the ATPase activity of the F₁-ATPase and is believed to function as a regulator of the ATP synthase. This inhibition by the ε subunit (ε inhibition) of F₁-ATPase from thermophilic Bacillus PS3 was analyzed by single molecule measurements. By using a mutant ε subunit deficient in ATP binding, reversible transitions between active and inactive states were observed. Analysis of pause and rotation durations showed that the ε inhibition takes a different path from the ADP-Mg inhibition. Furthermore, the addition of the mutant ε subunit to the α₃β₃γ sub-complex was found to facilitate recovery of the ATPase activity from the ADP-Mg inhibition. Thus, it was concluded that these two inhibitions are essentially exclusive of each other.