Fluorescent probes were attached to the single sulfhydryl residue on the isolated epsilon polypeptide of chloroplast coupling factor 1 (CF1), and the modified polypeptide was reconstituted with the epsilon-deficient enzyme. A binding stoichiometry of one epsilon polypeptide per CF1 was obtained. This stoichiometry corresponded to a maximum inhibition of the Ca2+-dependent ATPase activity of the enzyme induced by epsilon removal. Resonance energy transfer between the modified epsilon polypeptide and fluorescent probes attached to various other sites on the enzyme allowed distance measurements between these sites and the epsilon polypeptide. The epsilon-sulfhydryl is nearly equidistant from both the disulfide (23 A) and the dark-accessible sulfhydryl (26 A) of the gamma subunit. Measurement of the distance between epsilon and the light-accessible gamma-sulfhydryl was not possible due to an apparent exclusion of modified epsilon from epsilon-deficient enzyme after modification of the light-accessible site. The distances measured between epsilon and the nucleotide binding sites on the enzyme were 62, 66, and 49 A for sites 1, 2, and 3, respectively. These measurements place the epsilon subunit in close physical proximity to the sulfhydryl-containing domains of the gamma subunit and approximately 40 A from the membrane surface. Enzyme activity measurements also indicated a close association between the epsilon and gamma subunits: epsilon removal caused a marked increase in accessibility of the gamma-disulfide bond to thiol reagents and exposed a trypsin-sensitive site on the gamma subunit. Either disulfide bond reduction or trypsin cleavage of gamma significantly enhanced the Ca2+-ATPase activity of the epsilon-deficient enzyme. Thus, the epsilon and gamma polypeptides of coupling factor 1 are closely linked, both physically and functionally.