Although the reverse transcriptase (RT) of human immunodeficiency virus (HIV) uses human tRNA(3Lys) as a primer of viral genome DNA synthesis in vivo, HIV RT binds Escherichia coli glutamine tRNA and in vitro-made human lysine tRNA with nearly equivalent affinities. We show that HIV RT can use either tRNA(3Lys) or tRNA(2Gln) as a primer for DNA synthesis in vitro without the addition of any other host or viral proteins. E. coli tRNA(2Gln) can serve as a primer for HIV RT if a primer-binding site sequence complementary to the 3' end of tRNA(2Gln) is at the 3' end of the template. With this reduced template, the specificity of binding the proper tRNA is due to base-pairing between a bound tRNA to the primer-binding site of the viral RNA template rather than sequence-specific recognition of tRNA(3Lys) by RT. If an 8-nucleotide viral sequence 3' to the primer-binding site is included in the template, then addition of Zn2+ or Co2+ is required for tRNA(3Lys)-primed synthesis, and tRNA(2Gln) now fails to prime synthesis. The latter result implies that a template sequence adjacent to the primer-binding site and containing 6 nucleotides complementary to the anticodon loop of human tRNA(3Lys) plays an active role in tRNA discrimination.