Abstract A method for reconstitution of membrane proteins into unilamellar liposomes is described. The model enzyme was the F 0F 1ATP synthase from mitochondria when in complex or free from its inhibitor protein. The enzymes were first solubilized with either of two detergents, i.e., n-dodecyl-β- Dmaltoside or lauryldimethylamine oxide. After solubilization, the enzymes were passed through a column of Sepharose-AH using an ADP/sodium cholate selective elution buffer. The enzymes recovered from the column were subsequently passed through a centrifuge column of Sephadex G-50 fine. The eluate contained liposomes in which the F 0F 1complex (with and without inhibitor protein) had been reconstituted. The reconstituted enzymes were capable of hydrolyzing ATP with formation of electrochemical H +gradients. They also catalyzed the ATP–P iexchange reactions. Thus the F 0F 1complex which is formed by 18 subunits can be rapidly reconstituted into liposomes in a fully functional state. Moreover the data show that the interactions between the enzyme and its inhibitor protein are not perturbed in the reconstitution procedure.