Two sulfhydryl groups in the gamma subunit of solubilized chloroplast coupling factor 1 (CF1) are exposed by heat activating the enzyme. These two groups have been selectively labeled with [3H]-N-ethylmaleimide, N-[p-(2-benzoxazolyl)phenyl]maleimide (NBPM), and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM). Modifying these groups did not appreciably effect Ca2+-ATPase activity, the ability of the enzyme to bind quercetin and 1,N6-ethenoadenosine diphosphate (epsilonADP), or the ability of the enzyme to react with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1). Fluorescence resonance energy transfer was used to measure the distance from the sulfhydryl groups on the gamma subunit to the quercetin sites, the NBD-C1 reactive sites, and the tight nucleotide sites on CF1 using the donor-acceptor pairs NBPM-quercetin, NBPM-NBD, and epsilonADP-DDPM, respectively. The distance from the sulfhydryl groups to the quercetin sites was found to be less than 30 A, the distance to the NBD-C1 reactive was 34-41 A, and the distance to the tight nucleotide sites as greater than 40 A. A three-dimensional symmetrical model is proposed for the relative positions of sites on CF1.