Publisher Summary This chapter discusses the structure, synthesis, purification, and biogenesis of chloroplast adenosine triphosphatase (ATPase) (CF0–CF1). The isolation of the proton–ATPase complex from chloroplast is based on liberation of the complex from the membrane by detergent treatment. The proton–ATPase complex should be isolated from purified chloroplast membranes that are essentially free of the enzyme ribulose bisphosphate carboxylase. In biogenesis studies, the starting material is often intact chloroplasts that should be lysed and the membranes should be washed thoroughly prior to the isolation of CF0–CF1. The purified proton–ATPase complex can be stored at –70° for several months with a little loss of its specific activity. To obtain preparation of CFo–CF1 active in ATP–Pi exchange, the chloroplast membranes should be treated with 100 mM dithiothreitol for 30 min prior to the purification procedure. The ATP–Pi exchange and adenosine diphosphate (ADP)-phosphorylation activities of the enzyme are assayed after reconstitution of the enzyme into phospholipid vesicles. The chapter illustrates the electrotransfer of polypeptides from slab gels to nitrocellulose paper and immunodecoration by 125I-labeled protein. This technique is useful for identification of a given protein in sodium dodecyl sulfate (SDS) gels. Specific proteins can be detected and their quantity in crude cell extracts can be estimated.