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. It has recently been shown that the OmpA insertion into DOPC bilayers exhibits three kinetic phases with two membrane-bound intermediate forms (1). In the present study we sought to determine structural differences between the folding intermediates 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. Four different brominated stearoyl palmitoyl phos pha ti dyl chol ines (SPPC), with bromines in positions 4/5, 6/7, 9/10, and 11/12 were used to measure their effect on OmpA tryptophan fluorescence quenching during the time course of folding and membrane insertion. At 40°C and after 30 min or longer incubation, bilayers containing 6,7-diBrSPPC and 9,10-diBrSPPC were the most efficient quenchers of Trp-fluorescence. The next best quencher was 4,5-diBrSPPC followed by 11,12-diBrSPPC. In the first minutes of OmpA insertion differences between the different quenchers were much less expressed, with 11,12-diBrSPPC being a much more efficient quencher in the early than in the later phases of insertion. At 2 °C, 4,5- and 6,7-diBrSPPC were the most efficient quenchers even after long incubation times. A temperature jump from 2 to 37 °C converted the low-temperature into the high-temperature quenching profile, supporting the notion that the average Trp-position moves into a deeper location in the bilayer during membrane insertion of OmpA. (1) Kleinschmidt and Tamm, 1996, Biochemistry 35, 12993-13000.