The initiation of reverse transcription of human immunodeficiency virus type 1 (HIV-1) exclusively utilizes tRNA(Lys,3) as a primer. Previous studies have shown that HIV-1 could use alternative tRNAs, such as tRNA(Ile) or tRNA(Hls), to initiate reverse transcription only if the primer binding site (PBS) was made complementary to the 3' terminal 18 nucleotides of the cognate tRNA. However, upon in vitro culture, the viruses with a PBS complementary to the alternative tRNAs rapidly reverted to generate a PBS complementary to tRNA(Lys,3). To investigate the process of reversion, we have constructed defective proviral genomes that contain a PBS complementary to tRNA(Ile) or tRNA(Hls). The genomes contain the gene for xanthine-guanosine phosphoribosyl transferase (gpt) in place of env. Cotransfection of these proviral genomes with a plasmid-encoding vesicular stomatitis virus G protein (VSV-G) results in viruses that undergo a single round of HIV-1 infection; successful infections are scored as cells resistant to the drug mycophenolic acid. Using this single-round infection system, we demonstrated that HIV-1 with a PBS complementary to tRNA(Ile) or tRNA(His) is three- to fivefold less efficient in replication as measured by production of drug-resistant cell colonies compared to the wild-type virus. These viruses predominantly used the cognate tRNA as primer in their initial round of replication, although we did obtain a single cell colony in which the PBS was complementary to tRNA(Lys,3). Using an HIV-1 provirus with a PBS complementary to yeast tRNA(Phe), we established a single-round infection system in which the infectivity of this mutant HIV-1 relies on transfected yeast tRNA(Phe). The results of our studies suggest that the mechanism for selection of the tRNA primer for initiation of reverse transcription relies primarily on the complementarity between the tRNA primer and the PBS.