F 1 -ATPase, composed of α, β, γ, δ, and ɛ subunits, is a unique enzyme in terms of its rotational catalytic activity. The smallest unit showing this function is the α 3 β 3 γ complex. We have investigated the α 3 β 3 γɛ ΔC (ɛ ΔC , truncated ɛ) complex from thermophilic Bacillus PS3 (TF 1 ′, 360 kDa) in the solution state by using the combination of extensive deuteration, segmental-labeling, and CRINEPT (cross-correlated relaxation-enhanced polarization transfer) NMR. Well-resolved CRINEPT–HMQC (heteronuclear multiple-quantum correlation) spectra of partially 15 N-labeled TF 1 ′ were obtained for this huge and asymmetric protein complex. The spectrum of the C-terminal domain of the β subunit revealed that the open form of the β subunit in the TF 1 ′ complex is similar to that of the free β monomer. The open β subunit in the TF 1 ′ complex does not exhibit high affinity for nucleotides unlike the monomer, but this is in agreement with the results of single-molecule analysis of TF 1 α 3 β 3 γ. On the other hand, the closed form of the β subunit in the TF 1 ′ complex was shown to be distinct from that of the nucleotide-bound β monomer. This is consistent with a previous report that the closed form of the TF 1 β monomer could be a catalytically activated state. The loop between the N-terminal β-barrel and the central domain is highly flexible in the TF 1 ′ complex, in contrast to that in the α 3 β 3 hexamer, suggesting that it is affected by the presence of the γ subunit in this area.