Targeting and assembly of the Escherichia coli inner membrane protein leader peptidase (Lep) was studied using a homologous in vitro targeting/translocation assay. Assembly of full-length Lep was efficient in the co-translational presence of membrane vesicles and hardly occurred when membranes were added post-translationally. This is consistent with the signal recognition particle-dependent targeting of Lep. Crosslinking experiments showed that the hydrophilic region P1 of nascent membrane-inserted Lep 100-mer was in the vicinity of SecA and SecY, whereas the first transmembrane domain H1 was in the vicinity of YidC. These results suggested that YidC, together with the Sec translocase, functions in the assembly of Lep. YidC might be a more generic component in the assembly of inner membrane proteins.