The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs by the extension of a tRNA(Lys,3) positioned at an 18- nucleotide sequence in the RNA genome referred to as the primer-binding site (PBS). We have found that mutations within the PBS and a region upstream in U5, designated the A loop, influenced the selection of the tRNA primer used to initiate reverse transcription. Surprisingly, a proviral genome that contained a PBS and A loop complementary to tRNA(Pro) resulted in the generation of viruses that contained two PBSs within the same genome: one of the PBSs in the virus was complementary to tRNA(Lys,3) while the second PBS was complementary to tRNA(Ile), tRNA(Pro), or tRNA(Lys,)3. There were 14 nucleotides separating the two PBSs in the viral genome. In the current study, DNA encompassing U5 and the dual PBS complementary to the different tRNAs were amplified by PCR and exchanged for the corresponding region in an infectious HIV-1 clone, HXB2. Transfection of the different proviruses into cells resulted in the production of viruses that were infectious as determined by coculture with SupT1 cells. PCR was used to amplify the PBS regions from the different proviral DNAs followed by DNA sequencing of individual PCR clones. Proviruses containing the dual PBS complementary to tRNA(Lys,3) and tRNA(Ile) stably maintained the dual PBS complementary to both of these tRNAs following in vitro culture, although we noted consistent G-to-T and AA-to-GG substitutions in the 14-nucleotide region between the PBSs. The viruses derived from genomes that contained the dual PBS complementary to tRNA(Lys,3) and tRNA(Pro) also maintained both PBSs following in vitro culture; a single mutation was noted after in vitro culture in the 14-nucleotide region between the PBSs, which changed a consensus integration site (CA dinucleotide) prior to the PBS complementary to tRNA(Pro). In contrast, the proviral genomes containing the dual PBS complementary to tRNA(Lys,3) were not stable and reverted back to a single PBS complementary to tRNA(Lys,3). The results of our studies suggest that only the 5'-proximal PBS has been used to initiate reverse transcription. On the basis of our results, a mechanism is proposed for the generation of a dual PBS, which provides new insights into HIV-1 reverse transcription.