AbstractPowered by proton‐motive force, the inner membrane translocase AcrB is the engine of the AcrAB‐TolC efflux pump in Escherichia coli. As proton conduction in proteins occurs along hydrogen‐bonded networks of polar residues and water molecules, knowledge of the protein‐internal water distribution and water‐interacting residues allows drawing conclusions to possible pathways of proton conduction. Here, we report a series of 6× 50 ns independent molecular dynamics simulations of asymmetric AcrB embedded in a phospholipid/water environment. Simulating each monomer in its proposed protonation state, we calculated for each trans‐membrane domain the average water distribution, identified residues interacting with these waters and quantified each residue's frequency of water hydrogen bond contact. Combining this information we find three possible routes of proton transfer connecting a continuously hydrated region of known key residues in the TMD interior to bulk water by one cytoplasmic and up to three periplasm water channels in monomer B and A. We find that water access of the trans‐membrane domains is regulated by four groups of residues in a combination of side chain re‐orientations and shifts of trans‐membrane helices. Our findings support a proton release event via Arg971 during the C intermediate or in the transition to A, and proton uptake occurring in the A or B state or during a so far unknown intermediate in between B and C where cytoplasmic water access is still possible. Our simulations suggest experimentally testable hypotheses, which have not been investigated so far. Proteins 2011; © 2011 Wiley‐Liss, Inc.