Fluorescence resonance energy transfer measurements were used to construct spatial maps for the accessible sulfhydryl of the .gamma. subunit (dark site) and the essential tyrosine residue of the .beta. subunits relative to previously mapped sites on the H+-ATPase from [spinach] chloroplasts. The extent of energy transfer was measured between a coumarinylmaleimide derivative reacted covalently at the dark site and acceptor species selectively bound at the .gamma.-disulfide and the 3 nucleotide binding sites of the solubilized coupling factor complex. The nucleotide energy acceptor was 2''(3'')-(trinitrophenyl)ATP, and the .gamma.-disulfide site was labeled with fluoresceinylmaleimide. The dark-site sulfhydryl also was labeled with pyrenylmaleimide which served as an energy donor for 7-chloro-4-nitro-2,1,3-benzoxadiazole reacted at the .beta.-tyrosine sites. Similar measurements were also made with pyrenylmaleimide covalently attached to the .gamma.-sulfhydryl accessible only under energized conditions on the thylakoid membrane surface (light site). The observed transfer efficiencies indicate that the dark-site sulfhydryl is .apprx. 45 .ANG. from all 3 nucleotide sites and 41-46 .ANG. from the .gamma.-disulfide site. The average distances separating the essential .beta.-tyrosines and the light- and dark-site sulfhydryls are 38 and 42 .ANG., respectively. (In calculating these distances, random orientation of the donor-acceptor dipoles was assumed.) The results are consistent with a previously described structural model of the intact enzyme and can be used to gain insight into the overall structural organization of .alpha.-, .beta.- and .gamma.-polypeptides within the coupling factor.