The conserved residue Thr42 of ε-subunit of the chloroplast ATP synthase of maize (Zea mays L.) was substituted with Cys, Arg, and Ile, respectively, through site-directed mutagenesis. The over-expressed and refolded ε-proteins were purified by chromatography on DEAE-cellulose and FPLC on mono-Q column, which were as biologically active (inhibiting Ca2+-ATPase activity and blocking proton gate) as the native ε subunit isolated from chloroplasts. The εT42C and εT42R showed higher inhibitory activities on the soluble CF1(-ε) Ca2+-ATPase than the εWT. The εT42I inhibited the Ca2+-ATPase activity of soluble CF1 and restored photophosphorylation activity of membrane-bound CF1 deficient in ε the most efficiently. Far-ultraviolet CD spectra showed that the portions of α-helix and β-sheet structures of the three mutants were somewhat different from εWT. Thus the conserved residue Thr42 may be important for maintaining the structure and function of the ε-subunit and the basic functions of the ε-subunit as far as an inhibitor of Ca2+-ATPase and the proton gate are related. and Zhang-Lin Ni, Da-Fu Wang, Jia-Mian Wei.