The arrangement and functional role of the gamma subunit of the Escherichia coli F1ATPase (ECF1) has been probed by protease digestion and avidin-biotin labeling experiments using wild-type enzyme and four mutants, gamma S8C, gamma T106C, gamma S179C, and gamma V286C, respectively. Trypsin was found to cleave the gamma subunit at four sites, Arg70, Lys199, Lys201, and Lys212. Cleavage at these four sites did not greatly reduce the high ATPase activity of the enzyme that is obtained when the epsilon subunit is removed by the protease treatment. However, prolonged trypsin cleavage led to loss of inhibition by epsilon subunit added back to the trypsin-treated enzyme. Endoproteinase-Lys-C cleaves the gamma subunit of ECF1 at three of the four sites, i.e. Lys199, Lys201, and Lys212, but not at Arg70. The enzyme was activated by treatment with this protease because of degradation and release of the epsilon subunit, but added pure epsilon subunit still caused inhibition of ATPase activity. Therefore, cleavage at Arg70 by trypsin is responsible for the loss of response to the epsilon subunit inhibition. Biotin was reacted with Cys residues at positions 8, 106, 179, and 286 in different gamma subunit mutants and the accessibility of the biotin to avidin monitored in the intact ECF1 from the different mutants. Avidin was able to react with biotin when incorporated at position 106, not at 8, 179, or 286. The four trypsin cleavage sites, Arg70, Lys199, Lys201, and Lys212, as well as Thr106 are in regions of the gamma subunit predicted to be mainly beta-sheet and beta-turn structures.