The method of fluorescence energy transfer is used to measure the distance from the tight nucleotide binding sites to the 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reactive sites on solubilized spinach chloroplast coupling factor 1 (CF1). The fluorescent adenine nucleotide analogs 1,N-6-ethenoadenosine diphosphate and 1,N-6-ethenoadenylyl imidodiphosphate were used as donors and 4-nitrobenzo-2-oxa-1,3-diazole bound to a tyrosine group and to an amino group were used as acceptors of energy transfer. Using three different donor-acceptor pairs, the distance measured varied from 38 to 43 A assuming both donor sites are equidistant from the acceptor site. A model is proposed for the location of the tight nucleotide binding sites and the active site on the alpha and beta subunits of CF1.