This paper presents a study of the role of positive charge in the P(i) binding site of Escherichia coli ATP synthase, the enzyme responsible for ATP-driven proton extrusion and ATP synthesis by oxidative phosphorylation. Arginine residues are known to occur with high propensity in P(i) binding sites of proteins generally and in the P(i) binding site of the betaE catalytic site of ATP synthase specifically. Removal of natural betaArg-246 (betaR246A mutant) abrogates P(i) binding; restoration of P(i) binding was achieved by mutagenesis of either residue betaAsn-243 or alphaPhe-291 to Arg. Both residues are located in the P(i) binding site close to betaArg-246 in x-ray structures. Insertion of one extra Arg at beta-243 or alpha-291 in presence of betaArg-246 retained P(i) binding, but insertion of two extra Arg, at both positions simultaneously, abrogated it. Transition state stabilization was measured using phosphate analogs fluoroaluminate and fluoroscandium. Removal of betaArg-246 in betaR246A caused almost complete loss of transition state stabilization, but partial rescue was achieved in betaN243R/betaR246A and alphaF291R/betaR246A. BetaArg-243 or alphaArg-291 in presence of betaArg-246 was less effective; the combination of alphaF291R/betaN243R with natural betaArg-246 was just as detrimental as betaR246A. The data demonstrate that electrostatic interaction is an important component of initial P(i) binding in catalytic site betaE and later at the transition state complex. However, since none of the mutants showed significant function in growth tests, ATP-driven proton pumping, or ATPase activity assays, it is apparent that specific stereochemical interactions of catalytic site Arg residues are paramount.