The H(+)-ATPase (ATP synthase) from chloroplasts was isolated, purified and reconstituted into phosphatidylcholine/phosphatidic-acid liposomes. Liposomes prepared by reverse-phase evaporation were treated with various amounts of Triton X-100 and protein incorporation was studied at each step of the solubilization process. After detergent removal by SM2-Biobeads, the activities of the resulting proteoliposomes were measured indicating that the most efficient reconstitution was obtained by insertion of the protein into preformed, detergent-saturated liposomes. The conditions for the reconstitution were optimized with regard to ATP synthesis driven by an artificially generated delta pH/delta psi. An important benefit of the new reconstituted CF0F1 liposomes is the finding that the rate of ATP synthesis remains constant up to 10 s, indicating a low basal membrane permeability.