Substrate specificities of three viral replicative polymerases of different origins (HIV reverse transcriptase, hepatitis C virus RNA polymerase, and herpes virus DNA polymerase) towards 2'F-NTP were studied. Activated DNA, polyA-oligoU₆, and (2'F-A)₂₀-oligoU₆ complexes were used as templates. It was shown that all DNA polymerases studied incorporate 2'F-NMP into the 3'-end of primer-template complexes. HIV reverse transcriptase and herpes virus DNA polymerase can elongate synthesis with both dNTP and 2'F-NTP. Homopolymer (2'F-A)₂₀ can serve as a template for the polymerization of UTP as well as 2'F-UTP catalyzed by hepatitis C virus polymerase although with the efficacy about five-to tenfold lower compared to the natural primer-template complex. The pyrophosphorolysis reaction of 2'F-CMP residue on the 3'-end of the primer catalyzed with HIV reverse transcriptase proceeds with an efficiency that is two orders of magnitude less compared to the natural dNMP residues in the same system.