The presence of the HIV reverse transcriptase (RT) resistance mutation, M184V, induced by lamivudine and abacavir treatment results in increased tenofovir, adefovir and zidovudine susceptibility for HIV-1 with zidovudine-associated RT mutations in vitro. Treatment with oral prodrugs of tenofovir and adefovir has resulted in substantial HIV-1 RNA reductions in antiretroviral-experienced patient populations who have lamivudine- and zidovudine-resistant HIV-1. An enzymatic analysis was undertaken to elucidate the mechanisms of altered drug susceptibilities of HIV-1 containing zidovudine-associated mutations in the presence or absence of M184V. The inhibition constants (Ki) for the active metabolites of tenofovir, adefovir and zidovudine did not vary significantly between recombinant mutant and wild-type RT enzymes. Although increased removal of chain-terminating inhibitors by pyrophosphorolysis and ATP-dependent unblocking correlated with reduced susceptibility of viruses with zidovudine-associated mutations, a reduction in the removal of chain-terminators was not observed, which would explain the increased drug susceptibility of mutants containing M184V plus zidovudine-associated mutations. However, analyses of single-cycle processivity of the mutant RT enzymes on heteropolymeric RNA templates showed that all M184V-containing mutant RT enzymes were less processive than wild-type RT, most notably for mutants expressing both zidovudine-associated mutations and M184V. Similarly, the in vitro replication capacity of a mutant virus expressing a zidovudine-associated mutation and M184V was significantly reduced compared with wild-type virus. The observed decrease in enzymatic processivity of the M184V-expressing RT enzymes might result in decreased viral replication, which then might contribute to the increased drug susceptibility of HIV-1 expressing these RT mutations.