F sub(0)F sub(1) ATP synthase harnesses a transmembrane electrochemical gradient for the production of ATP. When operated in reverse, this multiprotein complex catalyzes ATP hydrolysis. In bacteria, the epsilon subunit is involved in regulating this ATPase activity. Also, epsilon is essential for coupling ATP hydrolysis (or synthesis) to proton translocation. The epsilon subunit consists of a beta sandwich and two C-terminal helices, alpha 1 and alpha 2. The protein can switch from a compact fold to an alternate conformation where alpha 1 and alpha 2 are separated, resulting in an extended structure. epsilon from the thermophile Bacillus PS3 (T epsilon ) binds ATP with high affinity such that this protein may function as an intracellular ATP level sensor. ATP binding to isolated T epsilon triggers a major conformational transition. Earlier data were interpreted in terms of an ATP + T epsilon sub(extended) arrow right ATP.T epsilon sub(compact) transition that may mimic aspects of the regulatory switching within F sub(0)F sub(1) (Yagi et al. (2007) Proc. Natl. Acad. Sci. U.S.A., 104, 11233-11238). In this work, we employ complementary biophysical techniques for examining the ATP-induced conformational switching of isolated T epsilon . CD spectroscopy confirmed the occurrence of a large-scale conformational transition upon ATP binding, consistent with the formation of stable helical structure. Hydrogen/deuterium exchange (HDX) mass spectrometry revealed that this transition is accompanied by a pronounced stabilization in the vicinity of the ATP-binding pocket. Surprisingly, dramatic stabilization is also seen in the beta 8- beta 9 region, which is remote from the site of ATP interaction. Analytical ultracentrifugation uncovered a previously unrecognized feature of T epsilon : a high propensity to undergo dimerization in the presence of ATP. Comparison with existing crystallography data strongly suggests that the unexpected beta 8- beta 9 HDX protection is due to newly formed protein-protein contacts. Hence, ATP binding to isolated T epsilon proceeds according to 2ATP + 2T epsilon sub(extended) arrow right (ATP.T epsilon sub(compact)) sub(2). Implications of this dimerization propensity for the possible role of T epsilon as an antibiotic target are discussed.