The human immunodeficiency virus (HIV) and other retroviruses show extensive genomic variation, which is primarily due to error-prone replication by the viral reverse transcriptase (RT) enzymes. RT errors include misincorporation with subsequent extension of the mismatched terminal base, and extension of realigned primer-template duplexes. Whereas both RT-mediated mechanisms have been extensively studied in vitro, almost no in vivo experiments have been performed. In this work, we analyzed the ability of HIV-1 RT to extend a misaligned tRNA(Lys3) primer in vivo. This tRNA binds with its 3'-terminal 18 nt to a complementary sequence in the viral genome, referred to as the primer-binding site (PBS). We constructed a series of mutant viral genomes with small insertions or deletions in the PBS sequence, resulting in misalignment of the tRNA primer. Extension of the misaligned primer did occur with reasonable efficiency for some of the mutants, resulting in reversion to the wild-type viral sequence. The infectivity and reversion frequency of the PBS mutants is therefore a measure of the efficiency of extending a misaligned primer in vivo. Using virion-derived primer-template complexes, we also measured the tRNA-priming efficiency in vitro. The combined results show that HIV-1 RT can elongate a misaligned primer and that the efficiency of primer extension is determined by the extent of the mismatch.