Summary The hydrocarbon core of a bilayer is normally a strict barrier to the passage of polar or charged solutes. We have discovered a novel class of cationic peptides that efficiently cross this barrier without causing bilayer permeabilization. These peptides are fundamentally different from other classes of membrane active peptides, and may hold the key to understanding how to bypass the barrier of the hydrocarbon core on demand. Yet, the determinants of this behavior are currently unknown. Here we will explore the physical chemical basis of peptide translocation by comprehensively examining rationally designed sequence variants of a known translocating peptide. The overarching hypothesis of this proposal is that translocation occurs only if the peptides have a specific translocation-enabling sequence motif and optimal hydrophobicity. Here we will test the hypothesis that translocation of peptides across bilayers requires optimal hydrophobicity that ensures a low steady state population in a membrane, and a specific sequence motif consisting of two arginines spaced by two hydrophobic residues, embedded in a hydrophobic sequence. We will determine the importance of these translocation-permissive properties by varying them independently by rational sequence modification and assessing the effect on (1) translocation across bilayers, (2) binding to bilayers, and (3) the structural response of the bilayer to the peptides.