HIV-1 virions contain approximately equal amounts of tRNA(Lys,3) and tRNA(Lys1,2), yet tRNA(Lys,3) has been found to be exclusively used for initiation of reverse transcription. Since previous studies have shown that even if the primer binding site (PBS) was mutated to be complementary to tRNA(Lys1,2), the virus did not stably use tRNA(Lys1,2) to initiate reverse transcription, the virus must have evolved a mechanism for the exclusive use of tRNA(Lys,3) to initiate reverse transcription. To investigate how HIV-1 discriminates tRNA(Lys1.2) from tRNA(Lys,3) for initiation of reverse transcription, two proviral genomes that contain nucleotide changes in U5 and a PBS to be complementary to regions of tRNA(Lys1,2) were constructed. One genome contains 5 [HXB2(L12-AC)] nucleotides while another contains 15 [HXB2(L12-ACgg)] nucleotides in U5 complementary to the anticodon region of tRNA(Lys1,2). Viruses derived from the transfection of the proviral genomes were infectious in SupT1 cells. Analysis of the endogenous reverse transcription reactions from viruses derived from HXB2 (L12-AC) and HXB2 (L12-ACgg) obtained from transfection revealed that both exclusively used tRNA(Lys1,2) to initiate reverse transcription. Following extensive in vitro culture, though, sequence analysis of proviral genomes revealed that while the virus derived from HXB2(L12-AC) stably maintained a PBS complementary to tRNA(Lys1,2), the virus derived from HXB2 (L12-ACgg) had reverted back to contain a PBS complementary to tRNA(Lys,3). RNA modeling of the U5-PBS of the genome from HXB2(L12-AC) supports the conclusion that the fine specificity for discrimination between tRNA(Lys,3) and tRNA(Lys1,2) for use as a primer for HIV-1 reverse transcription resides in the structure of the U5-PBS region of the viral genome.