Inhibition of human immunodeficiency virus type 1 (HIV-1) replication was demonstrated by using tat- and rev-directed antisense oligoribonucleotides 68 and 69 nucleotides in length. In this study, human T-lymphoid cells were transduced with a murine amphotropic retroviral vector containing a polymerase III-driven chimeric gene consisting of the human tRNAimet sequence and the short tat- and rev-directed antisense sequences that had been shown before to inhibit HIV-1 replication. Pools of transduced, G418-resistant human T-lymphoid Jurkat or CEM cells showed reduced replication of HIV-1 in the presence of antisense-containing chimeric transcripts, but not with sense sequence-containing transcripts. These results demonstrate that short inhibitory antisense RNA transcripts can be stably expressed endogenously using polymerase III promoters, which can reduce replication of HIV-1. The approach described in this work combines the advantages of short and, usually, synthetic oligonucleotides with the stable intracellular expression of inhibitory genes for HIV-1 in target cells. Considering the small size of the described chimeric polymerase III genes, it appears feasible to combine multiple antiviral genes with the currently available retroviral vectors as gene delivery systems.