The arginine-specific reagent phenylglyoxal inactivated the vacuolar H(+)-ATPase of red beet. Inactivation by phenylglyoxal followed pseudo-first-order kinetics and a double log plot of the t1/2 of inactivation versus phenylglyoxal concentration yielded a slope of 1.18. Neither inorganic anions nor DIDS protected from phenylglyoxal-mediated inactivation of the H(+)-ATPase. Indeed, Cl- stimulated the rate of phenylglyoxal-mediated H(+)-ATPase inactivation relative to SO4(2-). ATP, but not MgATP or ADP, protected from phenylglyoxal-mediated inactivation and inactivation resulted in a decrease in the Vmax of the H(+)-ATPase with little effect on the Km. Collectively, these results are consistent with phenylglyoxal-mediated inactivation of the vacuolar H(+)-ATPase resulting from modification of a single arginine residue in the catalytic nucleotide binding site of the vacuolar H(+)-ATPase. Stimulation of phenylglyoxal-mediated inactivation by Cl- indicates that exposure of the phenylglyoxal-sensitive functional arginine residue is enhanced in the presence of Cl-. The failure of MgATP to protect from phenylglyoxal inactivation suggests that ATP, rather than MgATP, binds directly to the catalytic site and that Mg2+ may act to promote catalysis subsequent to ATP binding.