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ATP-binding cassette (ABC) exporters transport substrates across biological membranes using ATP hydrolysis by a process that involves switching between inward- and outward-facing conformations. Most of the structural studies of ABC proteins have been performed with proteins in detergent micelles, locked in specific conformations and/or at low temperature. In this article, we present recent data from our laboratories where we studied the prototypical ABC exporter MsbA during ATP hydrolysis, at 37°C, reconstituted in a lipid bilayer. These studies were possible through the use of luminescence resonance energy transfer spectroscopy in MsbA reconstituted in nanodiscs. We found major differences between MsbA in these native-like conditions and in previous studies. These include a separation between the nucleotide-binding domains that was much smaller than previously thought, and a large fraction of molecules with associated nucleotide-binding domains in the nucleotide-free apo state. These studies stress the importance of studying membrane proteins in an environment that approaches physiological conditions.