The ε subunit of F F -ATPase/synthase (F F ) plays a crucial role in regulating F F activity. To understand the physiological significance of the ε subunit-mediated regulation of F F in Bacillus subtilis, we constructed and characterized a mutant harboring a deletion in the C-terminal regulatory domain of the ε subunit (ε ). Analyses using inverted membrane vesicles revealed that the ε mutation decreased ATPase activity and the ATP-dependent H -pumping activity of F F . To enhance the effects of ε mutation, this mutation was introduced into a ∆rrn8 strain harboring only two of the 10 rrn (rRNA) operons (∆rrn8 ε mutant strain). Interestingly, growth of the ∆rrn8 ε mutant stalled at late-exponential phase. During the stalled growth phase, the membrane potential of the ∆rrn8 ε mutant cells was significantly reduced, which led to a decrease in the cellular level of 70S ribosomes. The growth stalling was suppressed by adding glucose into the culture medium. Our findings suggest that the C-terminal region of the ε subunit is important for alleviating the temporal reduction in the membrane potential, by enhancing the ATP-dependent H -pumping activity of F F .