Pyrophosphorolysis, the removal of nucleoside chain-terminators by a pyrophosphate (PPi) acceptor molecule, and a similar mechanism (nucleotide-dependent chain-terminator removal) which uses ATP as an acceptor molecule have been proposed as mechanisms of zidovudine (AZT) resistance. Recombinant HIV-1 wild-type reverse transcriptase (RT) and a mutant RT enzyme containing the AZT/thymidine analog resistance mutations D67N/K70R/T215Y were analyzed for pyrophosphorolysis and nucleotide-dependent chain-terminator removal activities. Our results confirm that pyrophosphorolysis and nucleotide-dependent chain-terminator removal are potential mechanisms of AZT and d4T resistance. However, tenofovir is less efficiently removed by pyrophosphorolysis and by nucleotide-dependent mechanisms. These results are consistent with the minor changes in susceptibility to tenofovir of the AZT/thymidine analog-resistant HIV RT mutants and the corresponding resistance of these mutants to AZT. The inability to remove tenofovir efficiently by these mechanisms may contribute to the durability of the HIV RNA response observed in patients treated with the oral prodrug, tenofovir disoproxil fumarate.