The a subunit of F1F0 (F1F0-ATP synthase) is a highly hydrophobic protein with five putative transmembrane helices which plays a central role in H+-translocation coupled with ATP synthesis/hydrolysis. In the present paper, we show that the a subunit produced by the in vitro protease-free protein synthesis system (the PURE system) is integrated into a preformed F-0 a-less F1F0 complex in Escherichia coli membrane vesicles and liposomes. The resulting F1F0 has a H+-coupled ATP synthesis/hydrolysis activity that is approximately half that of the native F1F0. By using this procedure, we analysed five mutations of F1F0, where the conserved residues in the a subunit (Asn(90), Asp(112), Arg(169), Asn(173) and Gln(217)) were individually replaced with alanine. All of the mutant F(0)a subunits were successfully incorporated into F1F0 showing the advantage over conventional expression in E. coli by which three (N90A, D112A, and Q217A) mutant a subunits were not found in FIR,. The NI 73A mutant retained full activity and the mutants D112A and Q217A had weak, but detectable, activity. No activity was observed for the R169A and N90A mutants. Asn(90) is located in the middle of putative second transmembrane helix and likely to play an important role in H+-translocation. The present study exemplifies that the PURE system provides an alternative approach when in vivo expression of membranous components in protein complexes turns out to be difficult.