Escherichia coli F O F 1 ATP synthase, a rotary nanomachine, is composed of eight different subunits in a ␣ 3 ␤ 3 ␥␦ab 2 c 10 stoichiometry. Whereas F O F 1 has been studied in detail with regard to its structure and function, much less is known about how this multisubunit enzyme complex is assembled. Single-subunit atp deletion mutants are known to be arrested in assembly, thus leading to formation of partially assembled subcomplexes. To determine whether those subcomplexes are preserved in a stable standby mode, a time-delayed in vivo assembly system was developed. To establish this approach, we targeted the time-delayed assembly of membrane-integrated subunit a into preformed F O F 1 lacking subunit a (F O F 1 -a) which is known to form stable subcomplexes in vitro. Two expression systems (araBADp and T7p-laco) were adjusted to provide compatible, mutually independent, and sufficiently stringent induction and repression regimens. In detail, all structural atp genes except atpB (