The initiation of HIV-1 reverse transcription is primed by a cellular tRNA(Lys,3) molecule which is bound to a complementary sequence near the 5' end of the viral RNA genome designated as the primer-binding site (PBS). Recent studies have suggested that sequences upstream of the PBS within U5 consisting of a stretch of adenine nucleotides (referred to as the A-loop) might be important in the selection and positioning of tRNA(Lys,3) primer used to initiate reverse transcription. To further explore the role that the A-loop plays in reverse transcription, we have constructed proviral genomes in which the PBS was changed so as to be complementary to the 3'-terminal 18 nucleotides of tRNA(Ile), tRNA(Pro), or tRNA(Trp) [pHXB(Ile), pHXB(Pro), or pHXB(Trp), respectively]; a second set of proviral genomes was constructed which contained additional mutations so that the A-loop regions were complementary to the anticodon region of tRNA(Ile) [pHXB(Ile-AC)], tRNA(Pro) [pHXB(Pro-AC)], or tRNA(Trp) [pHXB(Trp-AC)]. Transfection of the proviruses into COS-1 cells followed by coculture with SupT1 cells resulted in production of infectious virus. PCR was used to amplify the PBS regions which were subcloned into M13mp18 followed by DNA sequence analysis. After short-term culture, the PBSs of proviruses derived from pHXB(Ile), pHXB(Pro), and pHXB(Trp) reverted to be complementary to tRNA(Lys,3). The PBSs of the viruses derived from pHXB(Ile-AC) also reverted to be complementary to tRNA(Lys,3); the A-loop region was still complementary to tRNA(Ile). In contrast, viruses derived from transfection of pHXB(Pro-AC) initially maintained a PBS complementary to tRNA(Pro). Upon extended culture, we identified proviruses which contained PBSs complementary to two additional tRNAs: tRNA(Ile) and tRNA(Lys,3). Furthermore, we found proviruses which contain two PBSs within the same genome: one complementary to tRNA(Lys,3) and a second complementary to tRNA(Pro) or tRNA(Ile). Viruses derived from transfection of pHXB(Trp-AC) were the most delayed in appearance following transfection. Analysis of the PBS revealed that early after transfection, the majority of the PBSs were complementary to tRNA(Trp). After further in vitro culture, proviruses were identified with a PBS complementary to a new tRNA, tRNA(Met). Finally, upon extended culture, the viruses derived from the transfection of pHXB(Ile-AC), pHXB(Pro-AC), and pHXB(Trp-AC) contained mutations upstream from the PBS in U5 that created a stretch of 3 adenine nucleotides. The results of these studies then highlight the flexibility that exists with respect to the selection of the tRNA primer used to initiate HIV-1 reverse transcription.