Several nucleotide analogues have been described as inhibitors of NS5B, the essential viral RNA-dependent RNA polymerase of hepatitis C virus. However, their precise mode of action remains poorly defined at the molecular level, much like the different steps of de novo initiation of viral RNA synthesis. Here, we show that before elongation, de novo RNA synthesis is made of at least two distinct kinetic phases, the creation of the first phosphodiester bond being the most efficient nucleotide incorporation event. We have studied 2'-O-methyl-GTP as an inhibitor of NS5B-directed RNA synthesis. As a nucleotide competitor of GTP in RNA synthesis, 2'-O-methyl-GTP is able to act as a chain terminator and inhibit RNA synthesis. Relative to GTP, we find that this analogue is strongly discriminated against at the initiation step ( approximately 150-fold) compared with approximately 2-fold at the elongation step. Interestingly, discrimination of the 2'-O-methyl-GTP at initiation is suppressed in a variant NS5B deleted in a subdomain critical for initiation (the "flap," encompassing amino acids 443-454), but not in P495L NS5B, which shows a selective alteration of transition from initiation to elongation. Our results demonstrate that the conformational change occurring between initiation and elongation is dependent on the allosteric GTP-binding site and relaxes nucleotide selectivity. RNA elongation may represent the most probable target of 2'-modified nucleotide analogues, because it is more permissive to inhibition than initiation.