Enfuvirtide (ENF) is the first in a new class of antiretroviral agents targeting the fusion process of the viral life cycle. ENF is a synthetic 36-amino acid peptide that binds to the HR-1 region of gp41 preventing fusion of viral and cellular membranes. With the introduction of ENF there are now four classes of antiretrovirals each with distinct and different resistance pathways. Resistance to ENF among subtype B HIV-1 isolates is associated with amino acid changes mainly in the HR-1 region, although other regions of envelope have also been implicated. To determine whether subtype C viruses developed resistance mutations similar to subtype B viruses, 11 subtype C and 4 subtype B viruses were passaged in the presence of increasing concentrations of ENF. The subtype C isolates showed varying levels of replication at 1 microg/ml ENF by day 18, but by day 29 all replicated efficiently at 10 microg/ml ENF. All subtype C isolates showed evidence of genotypic changes in gp41 HR-1 following exposure to ENF that included G36S/E/D, I37T, V38M/A/L/E, N/S42D, N43K, L45R/M, and A50T/V. Three subtype C viruses had compensatory changes in the HR-2 region, which corresponds to the ENF sequence, and two isolates had changes in the V3 region. Mutational patterns among the four subtype B viruses were similar to those for subtype C and those previously published in the literature. These data indicate that in vitro resistance to ENF develops rapidly among HIV-1 subtype C isolates. In general, mutational patterns for subtype C were similar to those described for subtype B, suggesting that the mechanism of action for ENF is similar for HIV-1 subtype B and C isolates.