In Gram-negative bacteria, phospholipids and lipopolysaccharide (LPS) form the outer membrane (OM), which is an asymmetric bilayer. The LPS molecule is assembled in the inner membrane (IM) from the lipid A core oligosaccharide and the O-antigen, which are synthesized in the cytoplasm. In Escherichia coli, seven essential proteins LptABCDEFG form the LPS transport system (Lpt), which exports the LPS molecule from the IM to the OM [1,2]. The LptB2FG complex is an ATP-binding cassette (ABC) exporter module and transports LPS from the outer leaflet of the IM to the periplasmic domain of LptC. Using pulsed dipolar electron spin resonance spectroscopy (PELDOR or DEER) [3] we characterized the conformational heterogeneity of the LptB2FG complex. We observed that in proteoliposomes the nucleotide-binding domains (NBDs) close upon ATP binding, which is consistent with previous results in detergent micelles [4]. This is coupled with an opening of the lateral gate formed by the transmembrane (TM) helices of LptF (TM1-F) and LptG (TM5-G). At the second lateral gate formed by TM5-F and TM1-G, short loops bound to these helices show a dynamic behavior. In both detergent micelles and proteoliposomes, the β-jellyroll domains of LptF and LptG show a rather limited interdomain flexibility. In summary, while the NBDs closes upon ATP binding as expected, the TMDs and the β-jellyroll domains show an asymmetric behavior, which might play an important role for the transport of LPS to LptC.