Another class of antiviral peptides that has been shown to act as fusion inhibitors are retrocyclins. These are synthetic, 18- residue, cyclic antimicrobial peptides that possess amino acid compositions and structures based on the theoretical GDC-0199 molecular weight product of human h-defensin pseudogenes. Retrocyclins have been found to inhibit HIV-1 infection in both in vitro and in ex vivo models and have been shown to exhibit antiviral activity against both R5 and X4 tropic clinical isolates of HIV-1. Retrocyclins have also retained their antiviral activity for over 1 week following application in non-human primates. Further, retrocyclins remain stable under acidic conditions, are resistant to boiling, and lack cytotoxic and proinflammatory activity at concentrations over 100 times their IC50. Because of its unique stability and safety, combined with its potent anti-HIV activity even in the presence of mucosal fluids, the retrocyclin analog RC-101 is currently being developed as an intravaginal microbicide to prevent sexually transmitted HIV-1. Retrocyclins prevent viral membrane fusion by binding the HR2 helix of gp41. Using multi-round, serial passaging of the HIV-1 R5 strain, BaL, in the presence of sub-inhibitory concentrations of the RC-101, we selected for partially-resistant mutants. In agreement with retrocyclins preventing gp41 activity, mutations in gp41 alone were shown to be sufficient for RC-101 resistance in pseudotyped viruses. These mutations identified in gp41 were Q66R and N126K, located in the HR1 and HR2 regions, respectively. Due to the cationic nature of these mutations, it was presumed that they might act to electrostatically repel the cationic RC-101 peptides. Here,wesought to delineate themechanismbywhichmutations in gp41 contribute to RC-101 resistance. Specifically, we determined that Q66R compromises gp41 fusion and entry kinetics, and that N126K behaves as a compensatory mutation to enhance gp41 activity in RC-101 resistance, as has been observed in resistance to ENF. This is the first time that mutations compromising gp41 activity, followedbyacompensatorymutation,havebeenobservedas a pattern of drug resistance used to evade a non-gp41-mimetic peptide. Additionally, we identified the activity of RC-101 against clinically relevant enfuvirtide-resistant mutants. We observed that while single HR1 mutations provide some degree of drug resistance, this resistance is specific for either RC- 101 or ENF. The V38A substitution, in particular, has been shown to be associated with resistance against multiple C-peptide type fusion inhibitors, yet we found V38A mutants NVP-BEZ235 remained susceptible to inhibition by RC-101.