In F(o)F(1) (F(o)F(1)-ATP synthase), proton translocation through F(o) drives rotation of the oligomer ring of F(o)-c subunits (c-ring) relative to F(o)-a. Previous reports have indicated that a conserved arginine residue in F(o)-a plays a critical role in the proton transfer at the F(o)-a/c-ring interface. Indeed, we show in the present study that thermophilic F(o)F(1s) with substitution of this arginine (aR169) to other residues cannot catalyse proton-coupled reactions. However, mutants with substitution of this arginine residue by a small (glycine, alanine, valine) or acidic (glutamate) residue mediate the passive proton translocation. This translocation requires an essential carboxy group of F(o)-c (cE56) since the second mutation (cE56Q) blocks the translocation. Rotation of the c-ring is not necessary because the same arginine mutants of the 'rotation-impossible' (c(10)-a)F(o)F(1), in which the c-ring and F(o)-a are fused to a single polypeptide, also exhibits the passive proton translocation. The mutant (aR169G/Q217R), in which the arginine residue is transferred to putatively the same topological position in the F(o)-a structure, can block the passive proton translocation. Thus the conserved arginine residue in F(o)-a ensures proton-coupled c-ring rotation by preventing a futile proton shortcut.