The precise mechanism by which the broad-spectrum anti-RNA virus agent ribavirin elicits its in vitro antiviral effect has remained a matter of debate. We have demonstrated that inhibition of cellular inosine monophosphate dehydrogenase (IMPDH) activity, and thus depletion of intracellular GTP pools, is the predominant mechanism by which ribavirin inhibits the replication of four flavi- and two paramyxoviruses (J Virol 79:1943-1947, 2005). As a consequence, induction of error catastrophe, which has been proposed as a mechanism by which ribavirin may elicit its anti-RNA virus activity, may be expected to have little, if any, impact on its antiviral effect. The flavivirus yellow fever virus (17D vaccine strain) was cultured for five consecutive passages in the presence of 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin), 5-ethynyl-1-beta-D-ribo-furanosylimidazole-4-carboxamide (EICAR) (the 5-ethynyl analog of ribavirin), or mycophenolic acid (MPA; a compound that exclusively inhibits IMPDH). The reduction in infectious virus yield brought about by ribavirin (as well as MPA and EICAR) was paralleled by a similar reduction in viral RNA yield; in case of error-prone replication, the infectious virus yield is expected to decrease significantly faster than the viral RNA yield. In addition, pre-extinction populations of the virus that has suffered a maximum impact of treatment with ribavirin did not accumulate an increased number of mutations. Very similar observations were obtained with EICAR and with MPA, a molecule that cannot be incorporated into viral RNA. These data thus allow us to conclude that the in vitro anti-yellow fever virus activity of ribavirin is independent of error-prone replication.