We identified a sequence embedded in the U3-R region of HIV-1 RNA that is highly complementary to human tRNA(3)(Lys). The free energy of annealing to tRNA(3)(Lys) is significantly lower for this sequence and the primer-binding site than for other viral sequences of similar length. The only interruption in complementarity is a 29-nucleotide segment inserted where a tRNA intron would be expected. The insert contains the TATA box for viral RNA transcription. The embedded sequence includes a 9-nucleotide segment previously reported to aid minus-strand transfer by binding the primer tRNA(3)(Lys). Reconstituting transfer in vitro, we show that including segments from the embedded sequence in the acceptor template, beyond the 9 nucleotides, further increases transfer efficiency. We propose that a gene encoding tRNA(3)(Lys) was incorporated during HIV-1 evolution and retained, largely intact, because of its roles in transcription and strand transfer.