In all retroviruses, reverse transcription is primed by a tRNA whose 3' end 18 nucleotides are complementary to the so called viral primer binding site. Previous work showed that reverse transcription of HIV-1 RNA is initiated by tRNA(3Lys). Using a variety of chemical and enzymatic structural probes, we investigated the interactions between HIV-1 RNA and its natural primer tRNA(3Lys). In addition to the predictable contacts between the viral primer binding site and the 3' end of tRNA(3Lys), a specific interaction takes place between an A-rich loop located upstream of the primer binding site region and the anticodon loop of tRNA(3Lys). This AAAA/Umcm5s2UUU loop-loop interaction is not observed when the natural primer is replaced by an in vitro synthesized tRNA(3Lys) transcript. Furthermore, dethiolation of the modified nucleotide mcm5s2U at position 34 of tRNA(3Lys) strongly destabilizes this interaction. Sequence and structure comparisons indicate that the primer/template loop-loop interaction is conserved in all HIV-1 isolates, and possibly also in HIV-2 and SIV.