The reverse transcriptase of the human immunodeficiency virus type 1 (HIV-1 RT) does not possess an exonucleolytic proofreading activity; however, previous studies have shown that this enzyme can excise incorporated chain-terminators in the presence of pyrophosphate or ATP. This type of reaction provides a plausible mechanism for HIV-1 resistance to several nucleoside analogue inhibitors. Here we studied the efficiency of pyrophosphorolysis in the context of mismatched nucleotides, and found that the removal of dCMP and dTMP opposite T is literally blocked. Thus, pyrophosphorolysis may not provide an alternative, universal proofreading mechanism, although excision of dGMP and the correct dAMP opposite T can occur with considerable efficiency. Site-specific footprinting experiments revealed that the 3' end of C:T- and T:T-mispaired primer strands is predominantly found in a post-translocational configuration, which prevents the removal of terminal nucleotides. In contrast, complexes containing G:T and A:T base pairs can exist in both post- and pre-translocational stages. Excision can only occur in the latter, which helps to explain the observed selectivity of the reaction. The efficiency of mismatch extensions does not appear to depend on pre-existing changes of the translocational equilibrium. However, footprints of complexes containing 3' penultimate mismatches suggest that the incorporation of the first nucleotide following the mispair can force the enzyme to slide backwards, which can inhibit ensuing polymerization events. The fact that misincorporated nucleotides can affect the precise positioning of RT provides a rational for the development of novel nucleoside analogue inhibitors that contain modifications in the base moiety.