Replication of the human immunodeficiency virus type 1 (HIV-1) and other retroviruses involves reverse transcription of the viral RNA genome into a double-stranded DNA. This reaction is primed by the cellular tRNA(3Lys) molecule, which binds to a complementary sequence in the viral genome, referred to as the primer-binding site (PBS). In order to study the specificity of primer usage, we constructed a set of HIV-1 mutants with altered PBS sites corresponding to other tRNA species (tRNA(Ile), tRNA(1,2Lys), tRNA(Phe), tRNA(Pro), tRNA(Trp)). These mutant viruses were able to replicate, although with delayed replication kinetics compared with wild-type HIV-1. Identification of the tRNA species associated with the genomic RNA demonstrated binding of tRNAs complementary to the new PBS sites. However, the occupancy of the mutant PBS sites by these new primers was reduced and correlated well with the replication potential of the mutant viruses. These results suggest that the PBS sequence is not sufficient for annealing of the tRNA primer. Upon prolonged culturing, all mutants reverted to the wild-type PBS(3Lys) sequence. Minor sequence changes in the nucleotides flanking the PBS site indicate that these reversions resulted from annealing of the wild-type tRNA(3Lys) primer onto the mutant PBS sites, followed by copying of part of the tRNA(3Lys) sequence during reverse transcription. Furthermore, the reversion efficiency of the different PBS mutants was found to correlate with their tRNA(Lys)3 binding capacity. A remarkable reversion pathway was observed for the PBSPro variant (PBSPro-->PBSIle-->PBSwt). This pathway can be explained by efficient base pairing of tRNA(Ile) to PBSPro, followed by annealing of tRNA(3Lys) onto the PBSIle intermediate. These results demonstrate that HIV-1 is dedicated to the tRNA(3Lys) primer and that factors other than the PBS sequence determine the selective primer usage of this retrovirus.