The insertion of inner membrane proteins in Escherichia coli occurs almost exclusively via the SecYEG pathway, while some membrane proteins require the membrane protein insertase YidC. In vitro analysis demonstrates that subunit a of the F(1)F(0) ATP synthase (F(0)a) is strictly dependent on Ffh, SecYEG and YidC for its membrane insertion but independent of the proton motive force. The insertion of the first transmembrane segment of F(0)a also depends on Ffh and SecYEG but not on YidC, whereas the insertion is strongly dependent on the proton motive force, unlike the full-length F(0)a protein. These data demonstrate an extensive role of YidC in the assembly of the F(0) sector of the F(1)F(0) ATP synthase.