Colicins are water-soluble toxins that, upon interaction with membranes, undergo a conformational change, insert, and form pores in them. Pore formation activity is localized in a bundle of 10 α-helices named the pore-forming domain (PFD). There is evidence that colicins attach to the membrane via a hydrophobic hairpin embedded in the core of the PFD. Two main models have been suggested for the membrane-bound state: penknife and umbrella, differing in regard to the orientation of the hydrophobic hairpin with respect to the membrane. The arrangement of the amphipathic helices has been described as either a compact three-dimensional structure or a two-dimensional array of loosely interacting helices on the membrane surface. Using molecular dynamics simulations with an implicit membrane model, we studied the structure and stability of the conformations proposed earlier for four colicins. We find that colicins are initially driven towards the membrane by electrostatic interactions between basic residues and the negatively charged membrane surface. They do not have a unique binding orientation, but in the predominant orientations the central hydrophobic hairpin is parallel to the membrane. In the inserted state, the estimated free energy tends to be lower for the compact arrangements of the amphipathic helix, but the more expanded ones are in better agreement with experimental distance distributions. The difference in energy between penknife and umbrella conformations is small enough for equilibrium to exist between them. Elongation of the hydrophobic hairpin helices and membrane thinning were found unable to produce stabilization of the transmembrane configuration of the hydrophobic hairpin. Proteins 2010. © 2010 Wiley-Liss, Inc.