Cellular tRNALys-3 serves as the primer for reverse transcription of human immunodeficiency virus, type 1 (HIV-1). tRNALys-3 interacts directly with HIV-1 reverse transcriptase, is packaged into viral particles and anneals to the primer-binding site (PBS) of the HIV-1 genome to initiate reverse transcription. Therefore, the priming step of reverse transcription is a potential target for antiviral strategies. We have developed a mutant tRNALys-3 derivative with mutations in the PBS-binding region such that priming specificity was re-directed to the highly conserved TAR stem-loop region. This mutant tRNA retains high-affinity binding to HIV-1 reverse transcriptase, viral encapsidation, and is able to prime at both the targeted TAR sequence and at the viral PBS. Constitutive expression of mutant tRNA in T-cells results in marked inhibition of HIV-1 replication, as determined by measurements of viral infectivity, syncytium formation, and p24 production. Inhibition of retroviral replication through interference with the normal process of priming constitutes a new anti-retroviral approach and also provides a novel tool for dissecting molecular aspects of priming.