During reverse transcription of the genomic RNA of human immunodeficiency virus type 1 (HIV-1) into double-stranded DNA, reverse transcriptase (RT) must accommodate RNA-RNA, DNA-RNA, RNA-DNA and DNA-DNA hybrids as primer-template. In this study, we examined extension of RNA-tRNA3Lys, and DNA-tRNA3Lys complexes by HIV-1 RT. When the 3' end of tRNA3Lys is annealed to oligoribonucleotides, tRNA3Lys, but not the complementary RNAs, is extended by HIV-1 RT, indicating that tRNA3Lys is efficiently used as primer and RNA as template. An opposite primer usage is observed when tRNA3Lys is annealed to complementary oligodeoxyribonucleotides. In this case, the oligodeoxyribonucleotides are efficiently used as primer and tRNA3Lys as template. This result indicates that the nature of nucleic acid bound to tRNA3Lys determines which strand of the RNA-tRNA3Lys and DNA-tRNA3Lys hybrids is extended by HIV-1 RT. When an oligoribonucleotide is annealed to an unmodified transcript of tRNA3Lys, both nucleic acids are extended by HIV-1 RT, indicating that specific selection of tRNA3Lys as primer requires the post-transcriptional modifications of tRNA3Lys.