All retroviral genomes contain a nucleotide sequence designated as the primer binding site (PBS) which is complementary to the tRNA used for initiation of reverse transcription. For human immunodeficiency virus type 1 (HIV-1), all naturally occurring genomes have a PBS complementary to tRNA₃^Lys. However, within HIV-1 virions, there are approximately equal amounts of tRNA₁^Lys, tRNA₂^Lys, and tRNA₃^Lys. We have used an endogenous reverse transcription-PCR technique specific for the tRNA species within isolated HIV-1 virions to demonstrate that in addition to tRNA₃^Lys, tRNA₁^Lys and tRNA₂^Lys could be used for initiation of HIV-1 reverse transcription. Using a single-round infection assay which employed an HIV-1 genome with a gpt gene encoding xanthine-guanine phosphoribosyl transferase in place of the env gene, we generated cell lines resistant to mycophenolic acid. Analysis of the U5-PBS from single-cell clones revealed PBS complementary to tRNA₃^Lys, not tRNA₁^Lys or tRNA₂^Lys. A mutant HIV-1 genome was then created which would favor the completion of reverse transcription with tRNA_1,2^Lys. Using this provirus in the complementation system, we again found only genomes with a PBS complementary to tRNA₃^Lys from proviral DNA isolated from gpt-resistant single-cell colonies. Finally, infection of cells with a mutant HIV genome with a PBS complementary to tRNA_1,2^Lys resulted in gpt- resistant cell colonies which contained integrated provirions with a PBS complementary to tRNA_1,2^Lys. The results of these studies suggest that the selection of tRNA₃^Lys for initiation of HIV-1 reverse transcription occurs both at the initiation and at a postinitiation step in reverse transcription prior to integration of the proviral DNA.