The H(+)-translocating F(0)F(1)-ATP synthase of Escherichia coli functions as a rotary motor, coupling the transmembrane movement of protons through F(0) to the synthesis of ATP by F(1). Although the epsilon subunit appears to be tightly associated with the gamma subunit in the central stalk region of the rotor assembly, several studies suggest that the C-terminal domain of epsilon can undergo significant conformational change as part of a regulatory process. Here we use disulfide cross-linking of substituted cysteines on functionally coupled ATP synthase to characterize interactions of epsilon with an F(0) component of the rotor (subunit c) and with an F(1) component of the stator (subunit beta). Oxidation of the engineered F(0)F(1) causes formation of two disulfide bonds, betaD380C-S108C epsilon and epsilonE31C-cQ42C, to give a beta-epsilon-c cross-linked product in high yield. The results demonstrate the ability of epsilon to span the central stalk region from the surface of the membrane (epsilon-c) to the bottom of F(1) (beta-epsilon) and suggest that the conformation detected here is distinct from both the "closed" state seen with isolated epsilon (Uhlin, U., Cox, G. B., and Guss, J. M. (1997) Structure 5, 1219-1230) and the "open" state seen in a complex with a truncated form of the gamma subunit (Rodgers, A. J., and Wilce, M. C. (2000) Nat. Struct. Biol. 7, 1051-1054). The kinetics of beta-epsilon and epsilon-c cross-linking were studied separately using F(0)F(1) containing one or the other matched cysteine pair. The rate of cross-linking at the epsilon/c (rotor/rotor) interface is not influenced by the type of nucleotide added. In contrast, the rate of beta-epsilon cross-linking is fastest under ATP hydrolysis conditions, intermediate with MgADP, and slowest with MgAMP-PNP. This is consistent with a regulatory role for a reversible beta/epsilon (stator/rotor) interaction that blocks rotation and inhibits catalysis. Furthermore, the rate of beta-epsilon cross-linking is much faster than that indicated by previous studies, allowing for the possibility of a rapid response to regulatory signals.