HIV-1 evolves rapidly, which is thought to result from one or more error-prone steps in the virus life cycle. Because HIV-1 reverse transcriptase (RT) does not possess 3'- to 5'-exonucleolytic proofreading activity and because RT has been shown to be error-prone in cell free systems, it should be an important contributor to the high rate of HIV-1 mutation. However, because RNA polymerase II (pol II) synthesizes viral RNA, it might also contribute significantly to HIV-1 mutagenesis. To assess the relative contributions of RT and RNA pol II to HIV-1 mutagenesis, a system was established to study the rate and nature of mutations in HIV-1 long terminal repeats (LTRs). Owing to the unique nature of replication at the ends of the viral genome, mutational analysis of retroviral LTRs provides an opportunity to evaluate the relative contribution of HIV-1 RT and RNA pol II to viral mutagenesis. Mutational analysis was performed on both LTRs of 215 proviruses, restricted to a single cycle of replication, employing single-stranded conformational polymorphism and DNA sequencing allowing direct identification of mutations in the absence of selection and within autologous viral sequences. A total of 21 independent mutations was identified. Ten mutations were observed in both LTRs, which could have been introduced by either RT or RNA pol II, whereas the other eleven mutations were only present in a single LTR and could only have been introduced by RT. This provides the first direct evidence that HIV-1 RT contributes significantly to HIV-1 mutagenesis and is likely to be the primary engine for HIV-1 mutagenesis. Moreover, mutations were observed at the U3-R border, but the nature of the mutations and their frequency differed from experiments performed using cell-free systems suggesting that other viral and/or cellular factors contribute to fidelity at the ends of the viral genome.