In Escherichia coli, signal recognition particle (SRP)-dependent targeting of inner membrane proteins has been described. In vitro cross-linking studies have demonstrated that short nascent chains exposing a highly hydrophobic targeting signal interact with the SRP. This SRP, assisted by its receptor, FtsY, mediates the transfer to a common translocation site in the inner membrane that contains SecA, SecG, and SecY. Here we describe a further in vitro reconstitution of SRP-mediated membrane insertion in which purified ribosome-nascent chain-SRP complexes are targeted to the purified SecYEG complex contained in proteoliposomes in a process that requires the SRP-receptor FtsY and GTP. We found that in this system SecA and ATP are dispensable for both the transfer of the nascent inner membrane protein FtsQ to SecY and its stable membrane insertion. Release of the SRP from nascent FtsQ also occurred in the absence of SecYEG complex indicating a functional interaction of FtsY with lipids. These data suggest that SRP/FtsY and SecB/SecA constitute distinct targeting routes.