We assessed the natural genotypic and phenotypic susceptibilities to enfuvirtide of 171 HIV group O (HIV-O) samples and 29 strains, respectively. The N42D resistance-associated mutation in the gp41 region was detected in 98% of cases. The phenotypic assay showed a wide range of baseline susceptibilities, with 50% inhibitory concentrations (IC(50)s) from 4 to 5,000 nM, a range similar to that reported for HIV-1 group M. Thus, despite the natural genotypic resistance conferred by the N42D signature mutation, HIV-O variants appear to be phenotypically susceptible. Enfuvirtide could therefore potentially be used in antiretroviral treatments for HIV-O-infected patients.