Primary CD4⁺ T lymphocytes, supporting in vitro human immunodeficiency virus type 1 (HIV-1) replication, are destined to die by apoptosis. We explored the initial molecular events that act upstream from mitochondrial dysfunction in CD4⁺ T lymphocytes exposed to the HIV-1_LAI strain. We tracked by immunofluorescence the cells expressing the p24 viral antigen and used Percoll density gradients to isolate a nonapoptotic CD4⁺ T-cell subset with a high inner mitochondrial transmembrane potential (ΔΨm) but no outer mitochondrial membrane (OMM) rupture. In most p24⁺ (but not bystander p24⁻) cells of this subset, the lysosomes were undergoing limited membrane permeabilization, allowing the lysosomal efflux of cathepsins (Cat) to the cytosol. This was also induced by HIV-1 isolates from infected patients. Using pepstatin A to inhibit Cat-D enzymatic activity and Cat-D small interfering RNA to silence the Cat-D gene, we demonstrate that once released into the cytosol, Cat-D induces the conformational change of Bax and its insertion into the OMM. Inhibition of Cat-D activity/expression also conferred a transient survival advantage upon productively HIV-1-infected cells, indicating that Cat-D is an early death factor. The transfection of activated CD4⁺ T lymphocytes with a Nef expression vector rapidly induced the permeabilization of lysosomes and the release of Cat-D, with these two events preceding OMM rupture. These results reveal a previously undocumented mechanism in which Nef acts as an internal cytopathic factor and strongly suggest that this viral protein may behave similarly in the context of productive HIV-1 infection in CD4⁺ T lymphocytes.