The ultimate goal of the research proposed here is to elucidate the mechanisms of ATP hydrolysis and ATP synthesis by the ATP synthase, an enzyme common to all energy transducing membranes. At present, the information obtained on the structure of the ATP synthases in mitochondria chloroplasts and bacterial plasma is fragmentary. The enzyme from all sources consists of an F1 portion, outside the bilayer, made up of five or in some cases six different subunits, including the inhibitor protein. The F1 is bound to a membrane intercalated part, the FO portion, which is made up of three different subunits in the bacterial and chloroplast enzyme but more in the mitochondrial enzyme. The 5 subunits of F1 in Escherichia coli and beef heart mitochondria are present in the stoichiometry of Alpha, 3; Beta, 3; Gamma, l; Delta, 1; Epsilon; 1. The F1 contains three active sites that can work independently but only slowly. In the presence of an excess of substrate to enzyme, the F1 ATPase functions cooperatively and hydrolyzes ATP by a factor of 2 thousand to a million times faster than in single-site catalysis. Our immediate goals are to examine whether the three catalytic sites are structurally and functionally the same or not as well as to locate the active sites in the protein and identify residues critical to the observed cooperativity between these sites. Other structural studies are planned, including experiments to identify subunits of FO that bind the F1; experiments on the orientation of F1 with respect to the membrane and experiments to characterize the environment of a critical carboxyl in the FO, i.e. the DCCD-binding glutamic acid (Asp 61 in subunit c of Escherichia coli). Longer term studies will include attempts to crystallize both F1 and FO and experiments on the conformation of F1 and FO, as well as estimation of functionally important conformational changes in the ATP synthase.