The replication in human peripheral blood mononuclear cells (PBMC) of unique HIV-1 that select tRNAHis or tRNALys1,2 for reverse transcription was compared to the wild-type virus that uses tRNA Lys,3. HIV-1 with only the primer-binding site (PBS) changed to be complementary to these alternative tRNAs Initially replicated more slowly than the wild-type virus in PBMC, although all viruses eventually reached equivalent growth as measured by p24 antigen. Viruses with only a PBS complementary to the 3′ terminal 18 nucleotides of tRNAHis or tRNALys1,2 reverted to use tRNALys3. HIV-1 with mutations in the U5-PBS to allow selection of tRNAHis and tRNALys1,2 following long-term growth in SupT1 cells were also evaluated for growth and PBS stability following replication in PBMC. Although both viruses initially grew slower than wild type, they maintained a PBS complementary to the starting tRNA and did not revert to the wild-type PBS after long-term culture in PBMC. Analysis of the U5-PBS regions following long-term culture in PBMC also revealed few changes from the starting sequences. The virus that stably used tRNAHis was less infectious than the wild type. In contrast, the virus that stably used tRNALys1,2 evolved to be as infectious as wild-type virus following extended culture in PBMC. The results of these studies highlight the impact of the host cell on the tRNA primer selection process and subsequent infectivity of HIV-1.