OmpA is predicted to form an eight stranded β-barrel in the outer membrane of E. coli. Unfolded OmpA spontaneously inserts into lipid bilayers upon rapid dilution of the denaturant urea. OmpA binds to lipid bilayers in two forms: It is “partially inserted, adsorbed” to bilayers in the gel phase and “fully inserted, native” when bound to bilayers in the liquid-crystalline phase (1). In the present study we sought to determine if the adsorbed form could be a folding intermediate during membrane insertion of OmpA. Kinetic refolding experiments were carried out in the presence of small unilamellar vesicles of DOPC at temperatures ranging from 2 to 40 °C. At all temperatures, the tryptophan fluorescence intensity maximum shifted rapidly from 350 nm (unfolded OmpA) to 333 nm (membrane-bound OmpA). The fluorescence intensity at 333 nm then grew with kinetics that were strongly temperature dependent: At high temperatures, the fluorescence intensity reached its maximum after several minutes following a single exponential time course; at low temperatures, the kinetics were more complex and at least an order of magnitude slower. Parallel SDS-PAGE and trypsin digestion experiments showed that within the first few hours only the adsorbed form was formed at low temperatures. Even at high temperatures, the time course of the conversion to the native form as determined by SDS-PAGE was slower than the tryptophan insertion kinetics. When adsorbed OmpA was subjected to a temperature shift from 2 to 40 °C, it was rapidly converted into the native form, as judged from the kinetics and fluorescence characteristics which precisely paralleled those of OmpA that was directly inserted at the higher temperature. We interpret these results as evidence that the adsorbed form can be converted into the native form and, therefore, is a folding intermediate during membrane insertion of OmpA. (1) Rodionova et al. , 1995, Biochemistry, 34, 1921-1929).