A new approach to studying membrane topology and permissive sites in membrane proteins expressed in Escherichia coli is described. The method is based on in vitro transposition of mini-Tn5 derivatives bearing dual pho-lac reporters [Alexeyev, M. F., and Winkler, H. H. (1999) J. Mol. Biol. 285, 1503-1513]. Two mini-Tn5 transposons, Tnpholac1 and Tnpholac2, were designed in such a way that their insertions can be converted either by restriction-ligation or by in vivo Cre-lox recombination into either sandwich reporter fusions or short amino acid (aa) tags (25 or 42 aa long). A set of 48 unique insertions in the gene coding for the Rickettsia prowazekii ATP/ADP translocase (Tlc) was generated using Tnpholac2. The topological information generated by these insertions was found in to be in good agreement with the existing topological model. Subsequently, these insertions were converted into both 25 and 42 aa tags, and the activity of the resulting mutants was determined. Also, site-directed mutagenesis was used to construct insertions in the loops, where no transposon hops were discovered. Of 13 extramembrane domains in Tlc, only 3 (loops 7, 10, and 13) were found to be permissive, which is in marked contrast to previous observations in the E. coli lactose permease (LacY), where most insertions in extramembrane domains were demonstrated to be permissive. The permissiveness of the insertion after I368 in TM IX lead us to reconsider the boundaries for this TM by placing I368 on the interface between TM IX and loop 10. Interestingly, the 25 aa insertions consistently have 2-fold higher activity than the corresponding 42 aa insertions, which is also in contrast with observations made on LacY. Finally, in this study we report, for the first time, the frequency of 10 base pair target duplications generated by in vitro Tn5 transposition.