The alpha 3 beta 3 gamma and alpha 3 beta 3 complexes of F1-ATPase from a thermophilic Bacillus PS3 were compared in terms of interaction with trinitrophenyl analogs of ATP and ADP (TNP-ATP and TNP-ADP) that differed from ATP and ADP and did not destabilize the alpha 3 beta 3 complex. The results of equilibrium dialysis show that the alpha 3 beta 3 gamma complex has a high affinity nucleotide binding site and several low affinity sites, whereas the alpha 3 beta 2 complex has only low affinity sites. This is also supported from analysis of spectral change induced by TNP-ADP, which in addition indicates that this high affinity site is located on the beta subunit. Single-site hydrolysis of substoichiometric amounts of TNP-ATP by the alpha 3 beta 3 gamma complex is accelerated by the chase addition of excess ATP, whereas that by the alpha 3 beta 3 complex is not. We further examined the complexes containing mutant beta subunits (Y341L, Y341A, and Y341C). Surprisingly, in spite of very weak affinity of the isolated mutant beta subunits to nucleotides (Odaka, M., Kaibara, C., Amano, T., Matsui, T., Muneyuki, E., Ogasawara, K, Yutani, K., and Yoshida, M. (1994) J. Biochem. (Tokyo) 115, 789-796), a high affinity TNP-ADP binding site is generated on the beta subunit in the mutant alpha 3 beta 3 gamma complexes where single-site TNP-ATP hydrolysis can occur. ATP concentrations required for the chase acceleration of the mutant complexes are higher than that of the wild-type complex. The mutant alpha 3 beta 3 complexes, on the contrary, catalyze single-site hydrolysis of TNP-ATP rather slowly, and there is no chase acceleration. Thus, the gamma subunit is responsible for the generation of a high affinity nucleotide binding site on the beta subunit in F1-ATPase where cooperative catalysis can proceed.