The γ and ε subunits of F(0)F(1)-ATP synthase from photosynthetic organisms display unique properties not found in other organisms. Although the γ subunit of both chloroplast and cyanobacterial F(0)F(1) contains an extra amino acid segment whose deletion results in a high ATP hydrolysis activity (Sunamura, E., Konno, H., Imashimizu-Kobayashi, M., Sugano, Y., and Hisabori, T. (2010) Plant Cell Physiol. 51, 855-865), its ε subunit strongly inhibits ATP hydrolysis activity. To understand the physiological significance of these phenomena, we studied mutant strains with (i) a C-terminally truncated ε (ε(ΔC)), (ii) γ lacking the inserted sequence (γ(Δ198-222)), and (iii) a double mutation of (i) and (ii) in Synechocystis sp. PCC 6803. Although thylakoid membranes from the ε(ΔC) strain showed higher ATP hydrolysis and lower ATP synthesis activities than those of the wild type, no significant difference was observed in growth rate and in intracellular ATP level both under light conditions and during light-dark cycles. However, both the ε(ΔC) and γ(Δ198-222) and the double mutant strains showed a lower intracellular ATP level and lower cell viability under prolonged dark incubation compared with the wild type. These data suggest that internal inhibition of ATP hydrolysis activity is very important for cyanobacteria that are exposed to prolonged dark adaptation and, in general, for the survival of photosynthetic organisms in an ever-changing environment.