Reverse transcription of the HIV-1 RNA genome is primed by the cellular tRNAlys3 molecule that anneals to a complementary sequence in the viral genome, the primer-binding site (PBS). Additional interactions between the tRNA primer and the viral RNA were proposed to play a role in reverse transcription. We recently identified an 8-nt element in the U5 region upstream of the PBS that is critical for initiation and processive elongation of reverse transcription. This motif was termed the primer activation signal (PAS), and is proposed to interact with the “antiPAS sequence” in the TΨC arm of tRNAlys3. In this study, we demonstrate that the efficiency of initiation of reverse transcription can be modulated by PAS mutations that strengthen or weaken the interaction with antiPAS. These results provide further evidence for a direct base-pairing interaction between the PAS in the viral RNA and the antiPAS in the tRNAlys3 molecule. A broad phylogenetic survey indicated that a PAS element is present in all retroviral RNA genomes, suggesting that the process of reverse transcription is regulated by a common mechanism in all retroviridae. It has proven very difficult to change the identity of the tRNA primer for HIV-1 reverse transcription by changing the PBS sequence. Using in vitro reverse transcription assays, we demonstrate that the identity of the priming tRNA species can be switched by simultaneous alteration of the PBS and PAS motifs to accommodate a new tRNA primer. These results indicate that the PAS–antiPAS interaction is important for both primer selection and efficient reverse transcription.