The structure of the isolated alpha subunit of F1-ATPase from the thermophilic Bacillus strain PS3 was probed using limited proteolysis by four different proteases, and the following results were obtained. 1) Distribution of 21 protease-cleaved sites is similar to that of the beta subunit of F1-ATPase (Tozawa, K., Odaka, M., Date, T., and Yoshida, M. (1992) J. Biol. Chem. 267, 16484-16490), thus providing experimental evidence for similar folding topology of the two subunits, and the locations of 11 water-exposed loop regions in the tertiary structure are predicted. 2) Most proteolytic peptides remain associated to maintain the gross structure of the alpha subunit and can reassociate each other after denaturing urea treatment. 3) However, the carboxyl-terminal peptides comprising approximately 80 residues (C1 peptides) are released from other peptide(s) during proteolysis, and those comprising approximately 105 residues (C2 peptides) are released during native polyacrylamide gel electrophoresis after proteolysis. 4) Inclusion of Mg-ATP in the native electrophoretic system prevents the release of the C2 peptide. Addition of Mg-ATP to the proteolysis mixtures results in an increase of the C2 peptide population and a decrease of the C1 peptide population. Thus, Mg-ATP induces a conformational change at the regions of C1 and C2 peptides of the alpha subunit. 5) Except for the trypsin-treated one, protease-treated alpha subunits are reconstitutable with the native beta subunit into the form of alpha 3 beta 3 complexes, which show significantly higher ATPase activities than the intact alpha 3 beta 3 complex. This activation is attributable to the cleavage of a peptide bond that produces C2 peptides. The carboxyl-terminal region of the alpha subunit is likely to be involved in the regulation of ATPase activity in F1-ATPase.