ABSTRACT There is considerable HIV-1 variation in patients. The extent of the variation is due to the high rate of viral replication, the high viral load, and the errors made during viral replication. Mutations can arise from errors made either by host DNA-dependent RNA polymerase II or by HIV-1 reverse transcriptase (RT), but the relative contributions of these two enzymes to the mutation rate are unknown. In addition, mutations in RT can affect its fidelity, but the effect of mutations in RT on the nature of the mutations that arise in vivo is poorly understood. We have developed an efficient system, based on existing technology, to analyze the mutations that arise in an HIV-1 vector in a single cycle of replication. A lacZ α reporter gene is used to identify viral DNAs that contain mutations which are analyzed by DNA sequencing. The forward mutation rate in this system is 1.4 × 10 ⁻⁵ mutations/bp/cycle, equivalent to the retroviral average. This rate is about 3-fold lower than previously reported for HIV-1 in vivo and is much lower than what has been reported for purified HIV-1 RT in vitro . Although the mutation rate was not affected by the orientation of lacZ α, the sites favored for mutations (hot spots) in lacZ α depended on which strand of lacZ α was present in the viral RNA. The pattern of hot spots seen in lacZ α in vivo did not match any of the published data obtained when purified RT was used to copy lacZ α in vitro .