Membrane proteins function naturally as imbedded in the lipid bilayers of cell membranes, but isolation into homogeneous and soluble preparations is needed for many biochemical studies. Detergents, which are used traditionally to extract and purify membrane proteins from cells, also remove most protein-associated lipid molecules as they disrupt the membranes. We have devised a detergent-free system to prepare native cell-membrane nanoparticles for biochemical analysis. In application to the membrane transporter AcrB, we demonstrate that these detergent-free nanoparticles are suitable for cryo-EM imaging at high resolution and that the natural lipid-bilayer structure so preserved is important for the functional integrity of AcrB. This nanoparticle system should be broadly applicable in membrane-protein research.