tRNA(3Lys), the primer for reverse transcriptase in human immunodeficiency virus type 1 (HIV-1), anneals to the primer binding site (PBS) in HIV-1 RNA. It has been shown that altering the PBS and U5 regions upstream of the PBS in HIV-1 so as to be complementary to sequences in tRNA(Met) or tRNA(His) will allow these tRNA species to be stably used as primers for reverse transcription. We have examined the replication of these mutant viruses in Sup-T1 cells. When Sup-T1 cells are infected by cocultivation with HIV-1-transfected 293T cells, viruses using tRNA(His) or tRNA(Met) are produced at rates that are approximately 1/10 or 1/100, respectively, of rates for wild-type virions that use tRNA(3Lys). When Sup-T1 cells are directly infected with equal amounts of these different viruses isolated from the culture supernatant of transfected 293T cells, virions using tRNA(Met) are produced at 1/100 the rate of wild-type viruses, and production of virions using tRNA(His) is not detected. Both wild-type and mutant virions selectively package tRNA(Lys) only, and examination of the ability of total viral RNA to prime reverse transcription in vitro indicates a >80% reduction in the annealing of tRNA(His) or tRNA(Met) to the mutant viral RNAs. PCR analysis of which of the three primer tRNAs is used indicates that only tRNA(3Lys) is detected as primer in wild-type virions and only tRNA(His) is detected as primer in virions containing a PBS complementary to tRNA(His), while the mutant viruses containing a PBS complementary to tRNA(Met) use both tRNA(Met) and tRNA(1,2Lys) as primer tRNAs.