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The MsbA protein is an essential ABC (ATP-binding-cassette) superfamily member in Gram-negative bacteria. This 65 kDa membrane protein is thought to function as a homodimeric ATP-dependent lipid translocase or flippase that transports lipid A from the inner to the outer leaflet of the cytoplasmic membrane. We have previously shown that purified MsbA from Escherichia coli displays high ATPase activity, and binds to lipids and lipid-like molecules, including lipid A, with affinity in the low micromolar range. Bacterial membrane vesicles isolated from E. coli overexpressing His6-tagged MsbA displayed ATP-dependent translocation of several fluorescently NBD (7-nitrobenz-2-oxa-1,3-diazole)-labelled phospholipid species. Purified MsbA was reconstituted into proteoliposomes of E. coli lipid and its ability to translocate NBD-labelled lipid derivatives was characterized. In this system, the protein displayed maximal lipid flippase activity of 7.7 nmol of lipid translocated per mg of protein over a 20 min period for an acyl chain-labelled PE (phosphatidylethanolamine) derivative. The protein showed the highest rates of flippase activity when reconstituted into an E. coli lipid mixture. Substantial flippase activity was also observed for a variety of other NBD-labelled phospholipids and glycolipids, including molecules labelled on either the headgroup or the acyl chain. Lipid flippase activity required ATP hydrolysis, and was dependent on the concentration of ATP and NBD–lipid. Translocation of NBD–PE was inhibited by the presence of the putative physiological substrate lipid A. The present paper represents the first report of a direct measurement of the lipid flippase activity of purified MsbA in a reconstituted system.