β-Arg-182 in Escherichia coli F1-ATPase (β-Arg-189 in bovine mitochondrial F1) is a residue which lies close to catalytic site bound nucleotide (Abrahams et al. (1994) Nature 370, 621-628). Here we investigated the role of this residue by characterizing two mutants, βR182Q and βR182K. Oxidative phosphorylation and steady-state ATPase activity of purified F1 were severely impaired by both mutations. Catalytic site nucleotide-binding parameters were measured using the fluorescence quench of β-Trp-331 that occurred upon nucleotide binding to purified F1 from βR182Q/βY331W and βR182K/βY331W double mutants. It was found that (a) β-Arg-182 interacts with the γ-phosphate of MgATP, particularly at catalytic sites 1 and 2, (b) β-Arg-182 has no functional interaction with the β-phosphate of MgADP or with the magnesium of the magnesium-nucleotide complex in the catalytic sites, and (c) β-Arg-182 is directly involved in the stabilization of the catalytic transition state. In these features the role of β-Arg-182 resembles that of another positively Charged residue in the catalytic site, the conserved lysine of the Walker A motif, β-Lys-155. A further role of β- Arg-182 is suggested, namely involvement in conformational change at the catalytic site β-α subunit interface that is required for multisite catalysis.