The mechanosensitive channel with large conductance (MscL) ofEscheri-chia coliis formed by a homopentameric assembly of MscL proteins. Here, we describe MscL biogenesis as determined usingin vivo approaches. Evi-dence is presented that MscL is targeted to the inner membrane via the sig-nal recognition particle (SRP) pathway, and is inserted into the lipid bilayer independently of the Sec machinery.