The subunit locations of each of the three nucleotide binding sites of soluble chloroplast coupling factor 1 have been studied with the photoaffinity label 3'-O-(4-benzoyl)benzoyl-ATP. This derivative is an effective inhibitor of ATPase activity. Photolysis of the radioactive label when bound to each of the three nucleotide sites on the coupling factor has been examined. For the nucleotide site that normally binds ADP very tightly, NaDodSO4/polyacrylamide gel electrophoresis after photolysis indicates that primarily the beta polypeptide chain is appreciably labeled (86%), although some labeling of the alpha polypeptide chain is found (14%). For the site that binds MgATP tightly, 97% of the radioactivity is found on the beta polypeptide chain. The alpha and beta polypeptide chains are labeled in approximately equal amounts when photolysis is carried out with the nucleotide analog bound to the third site.