The TAT protein transduction domain (PTD) of the human immunodeficiency virus (HIV-1) can cross cell membranes with unusual efficiency [1] and has many potential biotechnological applications.[2–4] Extant work has provided important clues to the molecular mechanism underlying the activity of this peptide, which consists of 11 amino acids, 8 of which are cationic and 6 of these are arginines. TAT PTD synthesized with d-amino acids enters cells as efficiently as the native form,[5] thereby indicating that the mechanism of transduction is receptor independent; this conclusion is consistent with recent results that suggest that the TAT PTD may enter cells through receptor-independent macropinocytosis.[6] Substitution of any of the PTD s cationic residues with neutral alanine decreases activity, while substitution of neutral residues has no effect.[5] This indicates the importance of electrostatic interactions between cationic TAT PTD and anionic phospholipid membranes. Recent work has shown that the physics of electrostatic interactions can drive a rich polymorphism of self-assembled structures that depend on parameters such as charge density [7, 8] and intrinsic membrane curvature.[9, 10] However, although arginine-rich polycations can enter cells, cationic polylysine cannot.[11] This shows that electrostatic interactions alone are insufficient for PTD activity and that the arginine residues play a specific, essential role. We use confocal microscopy and synchrotron X-ray scattering (SAXS) to study the interaction of the TAT PTD with model membranes at room temperature. We find that the transduction activity correlates with induction of negative Gaussian (“saddle-splay”) membrane curvature, which is topologically required for pore formation. Moreover, we show that the TAT PTD can drastically remodel vesicles into a porous bicontinuous phase with analogues in block-copolymer systems,[12–14] and we propose a geometric mechanism facilitated by both electrostatics and bidentate hydrogen bonding. The latter is possible for the TAT PTD but not for similarly cationic, nonarginated polypeptides. Cell membranes are composed of lipids that have fundamentally different interactions with cationic macroions such as TAT PTD. We examine representative model membranes composed of lipids with different charges and intrinsic curvatures: 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) have zwitterionic headgroups, while 1, 2-dioleoyl-sn-glycero-3-[phospho-l-serine](sodium salt)(DOPS) and 1, 2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)](sodium salt)(DOPG) have anionic headgroups; all have zero intrinsic curvature [15](C0= 0,“cylinder-shaped”) except for DOPE, which has negative intrinsic curvature (C0< 0,“cone-shaped”). When rhodamine-tagged TAT PTD (Rh-PTD) is applied to the exterior of giant unilamellar vesicles (GUVs, diameters of 5–30 μm) with low DOPE content (0 and 20%), rhodamine fluorescence is seen only outside the GUVs (Figure 1a), thereby indicating that the Rh-PTD has not crossed these membranes. However, when Rh-PTD is applied to GUVs with 40% DOPE content, the rhodamine intensity equilibrates across the membrane over tens of seconds (Figure 1b and c; see also the movie in the Supporting Information). This shows that Rh-PTD has crossed the GUV membranes, which remain intact (Figure 1 c). Thus, we see that the membrane transduction activity of Rh-PTD requires the presence of a threshold amount of DOPE in the membrane.