A novel HIV-1 genome that stably utilizes tRNA(His) rather than tRNA(Lys,3) to initiate reverse transcription was used to study features for the interaction between the tRNA and viral RNA genome. In addition to a primer binding site (PBS) complementary to tRNA(His), this virus contains a six-nucleotide sequence in U5 complementary to the anticodon-loop of tRNA(His) and three additional substitutions: U174-to-G, G181-to-A, and U200-to-C [HXB2(His-AC-GAC)]. Mutations in these three nucleotides resulted in viruses with three different genotypes: one group maintained a PBS complementary to tRNA(His) with restored G174A181C200 or G174A181U200 configurations, one group reverted to a PBS complementary to tRNA(Lys,3), and one group contained two or more PBSs complementary to different tRNAs on the same viral genome. Characterization of a previously identified virus with additional C152-to-A and C160-to-U substitutions [HXB2(His-AC-A152U160-GAC)] revealed that this virus maintained a PBS complementary to tRNA(His), whereas a mutant HXB2(His-AC-U152A160-GAC) reverted after culture to contain dual PBS complementary to tRNA(Lys,3) and tRNA(His), respectively. Our results demonstrate that regions in U5 act in concert with the PBS to promote use of the tRNA primer for initiation of reverse transcription. These results are discussed with respect to structural models for the U5-PBS interactions with tRNA.