Infection by human immunodeficiency virus type 1 (HIV-1) has been associated with increased cell death of both infected and bystander cells. The envelope glycoprotein complex appears to play an active role in HIV-induced death of bystander cells. We quantified cell-to-cell fusion, single cell death and membrane lipid mixing in cocultures of effector, HIV-1 envelope-expressing cells with peripheral blood mononuclear cells or purified CD4 T lymphocytes from HIV-negative donors, in the presence or the absence of the fusion inhibitor enfuvirtide (T-20, pentafuside, Fuzeon). T-20, which blocks gp41-dependent virus-cell fusion, showed a complete and dose-dependent inhibition of syncytium formation in cocultures of envelope-expressing cells with uninfected cells. Similarly, T-20 totally abrogated death of single bystander CD4 T cells with an IC50 of 0.04 microg/ml. Membrane lipid mixing, as a measure of interaction between envelope-expressing cells and CD4 cells, was also dose-dependently inhibited by T-20. Moreover, effector cells chronically infected with a T-20-resistant virus recovered the ability to induce bystander cell death in the presence of the drug, supporting the role of gp41 in single cell death. In conclusion, T-20 is able to protect CD4 T cells from envelope presentation with a dual effect: inhibition of virus replication and blockade of HIV-1 envelope-induced cell death of bystander CD4 T cells. Protection of cells prior to infection from HIV envelope-dependent bystander effect could lead to a better immune restoration of HIV-1-infected patients that are treated with T-20.