Human immunodeficiency virus type 1 (HIV-1) reverse transcription is primed by the cellular tRNA(3)(Lys) molecule that binds with its 3'-terminal 18 nucleotides to the fully complementary primer-binding site (PBS) on the viral RNA genome. Besides this complementarity, annealing of the primer may be stimulated by additional base-pairing interactions between other parts of the tRNA molecule and viral sequences flanking the PBS. According to the RNA secondary structure model of the HIV-1 leader region, part of the PBS sequence is involved in base pairing to form a small stem-loop structure, termed the U5-PBS hairpin. This hairpin may be involved in the process of reverse transcription. To study the role of the U5-PBS hairpin in the viral replication cycle, we introduced mutations in the U5 region that affect the stability of this structured RNA motif. Stabilization and destabilization of the hairpin significantly inhibited virus replication. Upon prolonged culturing of the virus mutant with the stabilized hairpin, revertant viruses were obtained with additional mutations that restore the thermodynamic stability of the U5-PBS hairpin. The thermodynamic stability of the U5-PBS hairpin apparently has to stay within narrow limits for efficient HIV-1 replication. Transient transfection experiments demonstrated that transcription of the proviral genomes, translation of the viral mRNAs, and assembly of the virions with a normal RNA content is not affected by the mutations within the U5-PBS hairpin. We show that stabilization of the hairpin reduced the amount of tRNA primer that is annealed to the PBS. Destabilization of the hairpin did not affect tRNA annealing, but the viral RNA-tRNA complex was less stable. These results suggest that the U5-PBS hairpin is involved in correct placement of the tRNA primer on the viral genome. The analysis of virus mutants and revertants and the RNA structure probing experiments presented in this study are consistent with the existence of the U5-PBS hairpin as predicted in the RNA secondary structure model.