Transmembrane potentials play important but poorly understood roles in many biological processes, including signal sequence-mediated protein translocation across bacterial membranes. In this study we applied Monte Carlo techniques to simulate the way the potential acts on a signal sequence. The simulations demonstrate that in the absence of a potential the signal sequence prefers insertion in both helical hairpin and transmembrane α-helical conformations. However, in the presence of a potential only the transmembrane α-helical conformation is the state of lowest energy for the signal sequence. From these results it is concluded that the membrane potential stabilizes the transmembrane orientation of a signal sequence, explaining the membrane potential dependence of preprotein translocation. © 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.