Residue Tyr-341 of the F1-ATPase beta subunit from a thermophilic Bacillus strain, PS3, was mutagenized to leucine, cysteine or alanine. Each of the mutated beta subunits was isolated and its affinity for ATP-Mg was examined by means of difference circular dichroism and differential titration calorimetry. The Kd values for ATP-Mg obtained were: beta Y341 (wild type), 0.015 mM; beta Y341L, 0.7 mM; beta Y341C and beta Y341A, > 3 mM. All the mutant beta subunits could be reconstituted into the alpha 3 beta 3 gamma complex with alpha and gamma subunits. The alpha 3 beta (mutant)3 gamma complexes hydrolyzed ATP with apparent Vmax values larger than that of the alpha 3 beta (WILD)3 gamma complex. The apparent Km values of the alpha 3 beta (mutant)3 gamma complexes increased in parallel with the Kd values for ATP-Mg of the isolated mutant beta subunits. These results indicate that residue beta Y341 is directly involved in the catalytic ATP-Mg binding and is a major Km-determining residue of F1-ATPase.