ATP synthase was isolated from beef heart mitochondria by extraction with N,N-bis-(3-~-gluconamidopropy1)deoxycholamide or by traditional cholate extraction. The enzyme was purified subsequently by ion-exchange and gel-permeation chromatographies in the presence of glycerol and the protease inhibitor diisopropylfluorophosphate. The ATP synthase consisted of 12-14 subunits and contained three tightly bound nucleotides. The co-reconstitution of crude or purified ATP synthase with monomeric bacteriorhodopsin by the method of detergent incubation of liposomes yielded proteoliposomes capable of light-driven ATP synthesis, as detected with a luciferase system for at least 30 min. The reaction was suppressed by the inhibitors oligomycin (> 90%) and dicyclohexylcarbodiimide (85%) and by the uncoupler carbonylcyanide-p-trifluormethoxyphenylhydrazone (>95%). The purified ATP synthase was apparently free of cytochrome impurities and of adenylate kinase activity, i.e. the enzyme exhibited light-driven ATP synthesis without the dark reaction. For the first time, this is demonstrated with purified ATP synthase from beef heart mitochondria, ATP synthase is a large membrane protein complex (M, = 500000), which plays a key role in the energy metabolism of most organisms [l-31. It couples the vectorial proton transport across a membrane to the formation or cleavage of the energy-rich metabolite, ATP. The enzyme consists of two subcomplexes, F, and F,, which can be separated in vitro. The larger subcomplex (Mr = 350000-400000), F, ATPase, is composed of five types of subunits designated a, , 8, y , 6and F in a stoichiometry of 3 : 3 : 1 : 1 : 1 [4-61. This watersoluble enzyme contains the catalytic and non-catalytic nucleotide-binding sites and catalyzes only hydrolysis of ATP if not bound to membranes. The smaller F,, part of the ATP synthase ( M r = 100000-150000) conducts the transfer of protons through the membranes. This process is driven by the electrochemical proton gradient supplied by the electrontransfer chains of respiration or photosynthesis. Depending on the organism, the F, moiety consists of three (bacteria, [7]), four (chloroplasts, [ 3 ] ) or 7-11 (mitochondria, [S-lo])