Oncoretroviral vectors were engineered to allow constitutive expression of an antisense RNA and the trans-activator of transcription (Tat)-inducible expression of a mRNA containing the trans-activation response (TAR) element, the Rev response element (RRE), and the efficient packaging signal (ψ^e) of human immunodeficiency virus-1 (HIV-1) RNA. Nuclear export of this mRNA by the regulator of expression of virion proteins (Rev) would allow its translation into wild type (WT) (MoTN-Ti-GE-Ri- Ter) or trans-dominant negative mutant (TDM) (MoTN-Ti-GmEm-Ri-Ter) Gag and Env proteins. Thus, the antisense RNA produced in a constitutive manner would ensure that even if there is leaky expression, no WT/TDM Gag or Env protein would be produced in the uninfected cells. If cells become infected by HIV-1, the antisense RNA would inhibit HIV-1 replication. Failure on the part of antisense RNA to inhibit virus replication would allow GE/GmEm mRNA production. The GE/GmEm mRNA would cause partial inhibition of HIV-1 replication as it contains the TAR, RRE, and ψ^e signal sequences. Translation of GmEm mRNA would give rise to TDM Gag and Env proteins, which would further decrease progeny virus infectivity. Tat- and Rev-inducibility was demonstrated in transfected HeLa and HeLa-Tev cells. Full-length WT/TDM Gag production was confirmed by Western blot analysis. Amphotropic vector particles were used to transduce a human CD4+ T-lymphoid cell line, and the stable transductants were challenged with HIV-1. Virus replication was better inhibited by the MoTN-Ti-GE-Ri-Ter vector than by the MoTN-Ti-GmEm-Ri-Ter vector. Inhibition of HIV-1 replication was also demonstrated in transduced CD4+ human peripheral blood T lymphocytes (PBLs). Moreover, our results suggest that cloning in the reverse transcriptional orientation must be avoided to prevent antisense RNA-mediated inhibition of transgene and endogenous gene expression.