Human immunodeficiency virus (HIV), like all retroviruses, requires a cellular tRNA as a primer for initiation of reverse transcription. In a previous study, we demonstrated that an HIV-1 with a primer binding site complementary to yeast tRNA(Phe) (psHIV-Phe) was not infectious unless yeast tRNA(Phe) was supplied in trans. This unique in vivo complementation system has now been used to define the elements of the tRNA required for HIV-1 replication. Mutant tRNA(Phe) with deletions in TΨC stem-loop, anticodon stem-loop or D stem-loop of the tRNA were generated and assessed for the capacity to rescue psHIV-Phe. Mutant tRNA(Phe) with disrupted TΨC stem-loop did not rescue psHIV-Phe. In contrast, a mutant tRNA(Phe) without the D stem-loop was fully functional for the rescue. The tRNA anticodon stem-loop region was found to be important for efficient complementation. The results of our studies demonstrate for the first time the importance of specific structural and sequence elements of the tRNA primer for HIV-1 reverse transcription and define new targets for interruption of HIV-1 replication.