Unfolded outer membrane protein A (OmpA) of E. coli spontaneously refolds and inserts into lipid bilayers upon dilution of denaturing urea. We have shown by time-resolved Trp fluorescence quenching that wild type OmpA, which contains 5 tryptophans, inserts into lipid bilayers via several structurally distinct folding intermediates [1]. To further dissect the folding pathway of OmpA, we have made by site directed mutagenesis and purified 5 different mutants, each containing a single Trp and 4 phenylalanines in the 5 Trp positions of the wild type protein. In Urea, each mutant contributed about 1/5th to the total fluorescence of the wild type protein (after subtracting minor tyrosine fluorescence contributions which were obtained from an additional Trp-less mutant). All mutants refolded in vitro into preformed vesicles of DOPC and, as judged by SDS-PAGE, their refolded state was indistinguishable from the native state of OmpA (cf. [2]). When incorporated into SUVs of DOPC, OmpA-Trp-7 exhibited a fluorescence intensity maximum at 327 nm, whereas the other mutants showed maxima in between 331 nm and 334 nm. Following the depth dependend evolution of Trp fluorescence quenching using 4 positional isomers of selectively sn-2-acyl-brominated phosphatidylcholines allowed us to calculate the distance of individual Trp residues from the bilayer center during refolding experiments. When monitored at 40 °C, which resolves the later steps of OmpA refolding [1, 2], Trp-7 moves from the membrane surface to about 10 Å from the bilayer center after 80 min. Similarly, Trp-15, Trp-57, Trp-102 and Trp-143 approached distances of 9 to 10.5 Å after 80 min. However, these latter Trps were found closer to the bilayer center, at 6 to 9 Å, 1 min after initiation of the refolding at 40 °C. [1] Kleinschmidt & Tamm, accompanying abstract. [2] Kleinschmidt & Tamm, 1996, Biochemistry 35, 12993-13000.