Autotransporters (AT) are outer membrane proteins (OMPs) in bacteria that contain both a channel-forming, b-barrel transmembrane domain (barrel-domain) and a passenger domain; that latter which is the secreted functional moiety. AT barrel-domains form a 12-stranded β-barrel in the outer membrane that facilitates translocation of the passenger domain to the bacterial cell surface. Following translocation, some AT passenger domains remain tethered to their b-domains, whereas the passenger domains of others are cleaved autoproteolytically and secreted into the extracellular milieu. While important advances in the field of autotransporter biogenesis have transpired over the past few years, the central issue of whether the barrel-domain forms the translocation channel for the passenger is still under debate. Furthermore, our understanding of how the autotransporter barrel-domain and β-barrel proteins, in general, fold and assemble into the outer membrane remains relatively limited. In this study, we used Pet from E. coli as a model autotransporter of the self-cleaving type, to investigate the biophysical properties of the Pet barrel-domain. Specifically, we used mutational analysis, circular dichroism and tryptophan fluorescence to monitor the folding of the Pet barrel-domain. All barrel-domains were produced in inclusion bodies and folded in vitro in both detergent micelles and liposomes. We found that the Pet barrel-domain folds rapidly, acquiring secondary and tertiary structure concurrently and provides evidence that the barrel-domain forms the translocation channel for the passenger domain. These results provide unique and detailed insights into the mechanism of barrel-domain folding and passenger domain secretion.