Higher plants possess two, distinct genes for the ATP synthase γ subunit, atpC1 and atpC2. In Arabidopsis, atpC1 is the predominant form, and atpC2 is only weakly expressed in photosynthetic tissues. There is no evidence that it plays any role in energy transduction. Indeed, mutants lacking atpC1 are incapable of photoautotrophic growth, while those lacking atpC2 have no noticeable phenotype. To elucidate the possible function of these orthologs, we analyzed mutants expressing exclusively atpC1 or atpC2 in Arabidopsis thaliana. In vivo chlorophyll fluorescence and electrochromic shift (ECS) analyses demonstrated that both atpC1 and atpC2 can function in ATP synthesis, though even under a strong promoter, the activity of atpC2-containing ATP synthase was low. However, we observed a striking difference in the regulation of ATP synthase containing the two orthologs. With atpC1, the ATP synthase was inactivated in the dark, likely via oxidation of the regulatory γ subunit thiols. ATP synthase containing exclusively atpC2 showed no decrease in activity even after extensive dark adaptation. We propose that atpC2 may function to catalyze low levels of ATP-driven proton translocation in the dark, when the bulk of ATP synthase is inactivated, maintaining sufficient transthylakoid proton gradient to drive protein translocation or other processes.