Cellular tRNA serves as the primer for reverse transcription of human immunodeficiency virus type 1 (HIV-1). tRNA interacts directly with HIV-1 reverse transcriptase (RT), is packaged into viral particles, and anneals to the primer-binding site (PBS) of the HIV-1 genome in order to initiate reverse transcription. Residue A58 of tRNA, which lies outside the PBS-complementary region, is posttranscriptionally methylated to form 1-methyladenosine 58 (M¹A58). This methylation is thought to serve as a pause signal for plus-strand strong-stop DNA synthesis during reverse transcription. However, formal proof that the methylation is necessary for the pausing of RT has not been obtained in vivo. In the present study, we investigated the role of tRNA residue A58 in the replication cycle of HIV-1 in living cells. We have developed a mutant tRNA derivative, tRNAA58U, in which A58 was replaced by U. This mutant tRNA was expressed in CEM cells. We demonstrate that the presence of M¹A58 is necessary for the appropriate termination of plus-strand strong-stop DNA synthesis and that the absence of M¹A58 allows RT to read the tRNA sequences beyond residue 58. In addition, we show that replacement of M¹A58 with U inhibits the replication of HIV-1 in vivo. These results highlight the importance of tRNA primer residue A58 in the reverse transcription process. Inhibition of reverse transcription with mutant tRNA primers constitutes a novel approach for therapeutic intervention against HIV-1.