Escherichia coli ATP synthase (F0F1) couples catalysis and proton transport through subunit rotation. The epsilon subunit, an endogenous inhibitor, lowers F-1-ATPase activity by decreasing the rotation speed and extending the duration of the inhibited state (Sekiya, M., Hosokawa, H., Nakanishi-Matsui, M., Al-Shawi, M. K., Nakamoto, R. K., and Futai, M. (2010) Single molecule behavior of inhibited and active states of Escherichia coli ATP synthase F-1 rotation. J. Biol. Chem. 285, 42058-42067). In this study, we constructed a series of epsilon subunits truncated successively from the carboxyl-terminal domain (helix 1/loop 2/helix 2) and examined their effects on rotational catalysis (ATPase activity, average rotation rate, and duration of inhibited state). As expected, the epsilon subunit lacking helix 2 caused about 1/2-fold reduced inhibition, and that without loop 2/helix 2 or helix 1/loop 2/helix 2 showed a further reduced effect. Substitution of epsilon Ser(108) in loop 2 and epsilon Tyr(114) in helix 2, which possibly interact with the beta and gamma subunits, respectively, decreased the inhibitory effect. These results suggest that the carboxyl-terminal domain of the epsilon subunit plays a pivotal role in the inhibition of F-1 rotation through interaction with other subunits.