The membrane-proximal external region (MPER; K(665)WASLWNWFNITNWLWYIK(683)) of the human immunodeficiency virus type 1 (HIV-1) gp41 ectodomain plays a critical role in envelope glycoprotein-mediated fusion. In addition, the epitopes of important neutralizing antibodies (2F5, Z13, and 4E10) and the sequence of the peptide fusion inhibitor T20 overlap this conserved region. The MPER has an unusually high percentage of tryptophan residues that likely contribute to the membrane-disrupting nature of the region, which is predicted to adopt an alpha-helical conformation on membrane contact. We have investigated the membrane-disruptive requirements for this region using a panel of mutants that replace most of the MPER with antibacterial, membrane-active peptides. The results demonstrate that the mutant Envs were processed, transported, and expressed on the cell surface similar to wild type. Some of the mutant Envs induced moderate levels of cell-cell fusion, demonstrating that the region can accommodate the substitution of proline-rich foreign peptides while retaining significant biological function. In contrast, the incorporation into and stability of the mutated Envs in virions was reduced, consistent with the severely impaired viral entry observed for all the mutants. These data suggest that both structural (for Env incorporation) and functional (membrane disruption) constraints may contribute to the highly conserved nature of this region.