Human immunodeficiency virus type 1 (HIV-1) selects a host cell tRNA as the primer for the initiation of reverse transcription. In a previous study, transport of the intact tRNA from the nucleus to the cytoplasm during tRNA biogenesis was shown to be a requirement for the selection of the tRNA primer by HIV-1. To further examine the importance of tRNA structure for transport and the selection of the primer, yeast tRNA(Phe) mutants were designed such that the native tRNA structure would be disrupted to various extents. The capacity of the mutant tRNA(Phe) to complement a defective HIV-1 provirus that relies on the expression of yeast tRNA(Phe) for infectivity was determined. We found a direct relationship between intact tRNA conformation and the capacity to be selected by HIV-1 for use in reverse transcription. tRNA(Phe) mutants that retained the capacity for nucleocytoplasmic transport, indicative of overall intact conformation, complemented the defective provirus. The mutant tRNAs were not aminoacylated, and the levels of complementation were lower than that for wild-type tRNA(Phe), which did undergo transport and aminoacylation. Taken together, these results demonstrate that HIV-1 primer selection is most dependent on a tRNA structure necessary for nucleocytoplasmic transport, consistent with primer selection occurring in the cytoplasm at or near the site of protein synthesis.