The H(+)/ATP ratio and the standard Gibbs free energy of ATP synthesis were determined with a new method using a chemiosmotic model system. The purified H(+)-translocating ATP synthase from chloroplasts was reconstituted into phosphatidylcholine/phosphatidic acid liposomes. During reconstitution, the internal phase was equilibrated with the reconstitution medium, and thereby the pH of the internal liposomal phase, pH(in), could be measured with a conventional glass electrode. The rates of ATP synthesis and hydrolysis were measured with the luciferin/luciferase assay after an acid-base transition at different [ATP]/([ADP][P(i)]) ratios as a function of deltapH, analysing the range from the ATP synthesis to the ATP hydrolysis direction and the deltapH at equilibrium, deltapH (eq) (zero net rate), was determined. The analysis of the [ATP]/([ADP][P(i)]) ratio as a function of deltapH (eq) and of the transmembrane electrochemical potential difference, delta micro approximately (H)(+) (eq), resulted in H(+)/ATP ratios of 3.9 +/- 0.2 at pH 8.45 and 4.0 +/- 0.3 at pH 8.05. The standard Gibbs free energies of ATP synthesis were determined to be 37 +/- 2 kJ/mol at pH 8.45 and 36 +/- 3 kJ/mol at pH 8.05.