Intracellular expression of genes that inhibit key steps in the human immunodeficiency virus (HIV-1) replicative cycle could offer an alternative therapy for AIDS treatment. One of these approaches involves the inhibition of env protein maturation through the expression of CD4 molecules with added exogenous sequences that promote their retention in the endoplasmic reticulum (ER). We have tested this strategy using a CD4 chimera (CD4epsilon10) containing an ER retention sequence derived from the TCR CD3-epsilon chain. Transfection of CD4epsilon10 in the human T cell line Jurkat made it resistant to infection with two different HIV-1 isolates, which was evaluated by measuring p24 antigen production, induction of apoptosis, and syncytia formation. Furthermore, polymerase chain reaction (PCR) analysis of genomic DNA showed no traces of the proviral HIV-1 genome in CD4epsilon10-transfected cells, suggesting it was not maintained latently in these cells. To facilitate the delivery of the CD4epsilon10 chimera to primary cells from AIDS patients, a Moloney-based retroviral vector was constructed that expresses CD4epsilon10 under the transcriptional control of the HIV-1 long terminal repeat (LTR) promoter. Transduction of the MT-2 human T cell line with this vector rendered it resistant to infection with HIV-1 by a process that involved the inhibition of gp160 proteolytic processing. Finally, transduction of the CD4epsilon10 chimera into T lymphoblasts derived from asymptomatic HIV-infected individuals demonstrated a protective effect, resulting in both an increased cellular proliferation rate and an increased percentage of CD4+ cells. These results suggest that it is feasible to use retroviral transduction of CD4epsilon10 as a gene therapy approach for AIDS treatment.