The high activity F₁-ATPase (the mutant α-C193S, γ-S107C, a ten histidine (His) tag inserted immediately downstream of the β initiation codon, α₃β₃γ subcomplex) of thermophilic Bacillus PS3 was purified from E.coli. JM103 Δ(uncB-uncD)，in which the majority of F₁-ATPase genes have been eliminated. It was found that the enzyme hydrolyzed ATP more efficiently than previous papers. During the F₁-ATPase hydrolyzing ATP, the rotary rate of the fluorescent actin filament attached to γ subunit of F₁-ATPase was about one times faster than that of previous papers in the similar conditions.