Escherichia coli F(O)F(1) ATP synthase, a rotary nanomachine, is composed of eight different subunits in a α3β3γδεab2c10 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 (encoding subunit a) were expressed under the control of araBADp and induced by arabinose. Following synthesis of F(O)F(1)-a during growth, expression was repressed by glucose/d-fucose, and degradation of atp mRNA controlled by real-time reverse transcription-PCR. A time-delayed expression of atpB under T7p-laco control was subsequently induced in trans by addition of isopropyl-β-d-thiogalactopyranoside. Formation of fully assembled, and functional, F(O)F(1) complexes was verified. This demonstrates that all subunits of F(O)F(1)-a remain in a stable preformed state capable to integrate subunit a as the last subunit. The results reveal that the approach presented here can be applied as a general method to study the assembly of heteromultimeric protein complexes in vivo.