The mutation of serine-174 to phenylalanine that causes a defect in the Escherichia coli F1-ATPase beta-subunit is suppressed by further mutations; Gly-149 to Ser, Ala-295 to Thr, Ala-295 to Pro, or Leu-400 to Gln (Miki, J., Fujiwara, K., Tsuda, M., Tsuchiya, T. and Kanazawa, H. (1990) J. Biol. Chem. 265, 21567-21572). We analyzed the effects of these second site mutations and of a newly identified Asn-158 to Tyr mutation on the activities of the ATPase without the original Ser-174 to Phe mutation. The beta-subunit with each amino acid replacement was expressed in the mutant strain JP17, which does not have a beta-subunit. Cells transformed with the plasmid carrying Ala-295 to Pro mutation alone did not grow on minimal medium agar supplemented with succinate as the sole carbon source, and showed 3% of the wild-type ATPase activity, suggesting that this mutation caused structural alterations affecting the catalytic function of the enzyme. Conversely transformants with other mutations grew well and had higher ATPase activities, suggesting that these mutations did not cause extensive structural alterations. From the transformants with the plasmid carrying the Ala-295 to Pro mutation, seven revertants capable of cell growth on succinate plates were isolated and reversion mutations were identified at residues 140, 159, 166, 171, 172 and 184 of the beta-subunits. The results suggested that Ser-174 and Ala-295 do not necessarily interact directly, but that the regions including these suppression mutation sites close to Ser-174, and Ala-295 interact with each other for the proper functioning of the ATPase. The ternary structure of the region surrounded by the residues which were identified as the reversion mutation sites for Ser-174 to Phe and Ala-295 to Pro mutations is important for the catalytic function of this enzyme.