Human tRNA3(Lys) is used by HIV virus as a primer for the reverse transcription of its genome. The 18 nucleotides at the 3'-end of the tRNA3(Lys) are hybridized to a complementary sequence of the viral RNA called the primer-binding site. A screen against the human tRNA3(Lys) over a peptide library designed to target RNA has been performed. Of the 175 hexapeptides tested, three were found to bind to the d-stem of tRNA3(Lys). Alanine-scanning was used to define the determinants of the interaction between the peptides and tRNA3(Lys). They also bind to two other tested tRNAs, also at the level of the d-stem and loop, although the nucleotide sequence of the stem differs in one of them. These short peptides thus recognize specific structural features within the d-stem and loop of tRNAs. Associated with other pharmacophores, they could be useful to design optimized ligands targeting specific tRNAs such as retroviral replication primers.