Abasic (AP) sites pose a potential danger to HIV-1 replication. HIV-1 RT has been shown to preferentially incorporate purines opposite an AP site, and subsequently extend from the lesion. While it is clear that AP sites are bypassed inefficiently and are major sites of RT pausing, detailed kinetic analysis of the relative contributions of both the incorporation and the pyrophosphorolytic reactions in translesion synthesis by HIV-RT is still lacking. Investigation of the molecular basis of these processes is important, in light of the fact that HIV-1 RT is the major target for anti-HIV chemotherapy, and its low fidelity is an essential determinant of the extraordinary genetic variability of HIV-1, which is important for the appearance of mutant viruses resistant to chemotherapy. Here, we analyzed the effects of the presence of an AP site on the template strand on the catalytic properties of the DNA-dependent polymerization reaction as well as on the phosphorolytic activity of HIV-1 RT, in the presence of deoxy-, dideoxy,- and ribonucleotides. We find that AP sites can substantially influence the substrate specificity of HIV-1 RT and that pyrophosphorolysis plays a significant role in determining the ability of HIV-1 RT to (mis)incorporate nucleotides Proteins 2008. © 2007 Wiley-Liss, Inc.