The ε subunits of several bacterial F1-ATPases bind ATP. ATP binding to the ε subunit has been shown to be involved in the regulation of F1-ATPase from thermophilic Bacillus sp. PS3 (TF1). We previously reported that the dissociation constant for ATP of wild-type ε subunit of TF1 at 25 °C is 4.3 μM by measuring changes in the fluorescence of the dye attached to the ε subunit (Kato, S. et al. (2007) J. Biol. Chem. 282, 37618). However, we have recently noticed that this varies with the dye used. In this report, to determine the affinity for ATP under label-free conditions, we have measured the competitive displacement of 2'(3')-O-N'-methylaniloyl-aminoadenosine-5'-triphosphate (Mant-ATP), a fluorescent analog of ATP, by ATP. The dissociation constant for ATP of wild-type ε subunit of TF1 at 25 °C was determined to be 0.29 μM, which is one order of magnitude higher affinity than previously reported values.