All known strains of HIV-1 use host tRNA(Lys3) as a primer for initiating reverse transcription. To understand why HIV has evolved to select this specific tRNA, we have examined potential secondary structures and found that human tRNA(Lys3) can adopt an alternative "slingshot" conformation that is almost isoenergetic to the conventional cloverleaf structure. We hypothesize that HIV-1 exploits the slingshot conformer in tRNA selection and formation of the initiation complex. This prediction is supported by two experimental tests. First, an in vitro transcribed mutant tRNA(Lys3) engineered to favor the slingshot promotes initiation complex formation more efficiently than wild-type tRNA(Lys3). Second, an examination of all known human tRNAs reveals that only tRNA(Glu) can also fold into a slingshot conformation with favorable energy, leading to the prediction that viruses engineered to use tRNA(Glu) should resist reversion to wild-type. The stability of these viruses is indeed much greater than that of viruses engineered to use other primers such as tRNA(His) or tRNA(Met). These results support a novel selection mechanism that explains why HIV-1 has evolved to preferentially utilize a specific tRNA for replication. They also provide a rationale for the development of new therapeutic targets for the design of drugs to inhibit HIV replication.