All naturally occurring human immune deficiency viruses (HIV-1) select and use tRNALys,3 as the primer for reverse transcription. Studies to elucidate the mechanism of tRNA selection from the intracellular milieu have been hampered due to the difficulties in manipulating the endogenous levels of tRNALys,3. We have previously described a mutant HIV-1 with a primer binding site (PBS) complementary to yeast tRNAPhe (psHIV-Phe) that relies on transfection of yeast tRNAPhe for infectivity. To more accurately recapitulate the selection process, a cDNA was designed for the intracellular expression of the yeast tRNAPhe. Increasing amounts of the plasmid encoding tRNAPhe resulted in a corresponding increase in levels of yeast tRNAPhe in the cell. The yeast tRNAPhe isolated from cells transfected with the cDNA for yeast tRNAPhe, or in the cell lines expressing yeast tRNAPhe, were aminoacylated, indicating that the expressed yeast tRNAPhe was incorporated into tRNA biogenesis pathways and translation. Increasing the cytoplasmic levels of tRNAPhe resulted in increased encapsidation of tRNAPhe in viruses with a PBS complementary to tRNAPhe (psHIV-Phe) or tRNA Lys,3 (wild-type HIV-1). Production of infectious psHIV-Phe was dependent on the amount of cotransfected tRNAPhe cDNA. Increasing amounts of plasmids encoding yeast tRNAPhe produced an increase of infectious psHIV-Phe that plateaued at a level lower than that from the transfection of the wild-type genome, which uses tRNALys,3 as the primer for reverse transcription. Cell lines were generated that expressed yeast tRNAPhe at levels approximately 0.1% of that for tRNA Lys,3. Even with this reduced level of yeast tRNAPhe, the cell lines complemented psHIV-Phe over background levels. The results of these studies demonstrate that intracellular levels of primer tRNA can have a direct effect on HIV-1 infectivity and further support the role for PBS-tRNA complementarity in the primer selection process. © 2003 Elsevier Science (USA). All rights reserved.