Antibody engagement with the membrane-proximal external region (MPER) of the envelopeglycoprotein (Env) of HIV-1 constitutes a distinctive molecular recognition phenomenon,the full appreciation of which is crucial for understanding the mechanisms that underlie the broadneutralization of the virus. Recognition of the HIV-1 Env antigen seems to depend on two specificfeatures developed by antibodies with MPER specificity: (i) a large cavity at the antigen-bindingsite that holds the epitope amphipathic helix; and (ii) a membrane-accommodating Fab surfacethat engages with viral phospholipids. Thus, besides the main Fab–peptide interaction, molecularrecognition of MPER depends on semi-specific (electrostatic and hydrophobic) interactions withmembranes and, reportedly, on specific binding to the phospholipid head groups. Here, based onavailable cryo-EM structures of Fab–Env complexes of the anti-MPER antibody 10E8, we soughtto delineate the functional antibody–membrane interface using as the defining criterion the neutralizationpotency and binding affinity improvements induced by Arg substitutions. This rational,Arg-based mutagenesis strategy revealed the position-dependent contribution of electrostatic interactionsupon inclusion of Arg-s at the CDR1, CDR2 or FR3 of the Fab light chain. Moreover, thecontribution of the most effective Arg-s increased the potency enhancement induced by inclusionof a hydrophobic-at-interface Phe at position 100c of the heavy chain CDR3. In combination, thepotency and affinity improvements by Arg residues delineated a protein–membrane interaction site,whose surface and position support a possible mechanism of action for 10E8-induced neutralization.Functional delineation of membrane-interacting patches could open new lines of research to optimizeantibodies of therapeutic interest that target integral membrane epitopes.