Fluorescence resonance energy transfer was used to measure the distances between three nucleotide binding sites on solubilized chloroplast coupling factor from spinach and between each nucleotide site and two tyrosine residues which are important for catalytic activity. The nucleotide energy donor was 1,N6-ethenoadenosine di- or triphosphate, and the nucleotide energy acceptor was 2'(3')-(trinitrophenyl)adenosine diphosphate. The tyrosine residues were specifically labeled with 7-chloro-4-nitro-2,1,3-benzoxadiazole, which served as an energy acceptor. The results obtained indicate the three nucleotide binding sites form a triangle with sides of 44, 48, and 36 A. (The assumption has been made in calculating these distances that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime.) Two of the nucleotide sites are approximately equidistant from each of the two tyrosines: one of the nucleotide sites is about 37 A and the other about 41 A from each tyrosine. The third nucleotide site is about 41 A from one of the tyrosines and greater than or equal to 41 A from the other tyrosine.