It is not yet clear to what extent depletion of intracellular GTP pools contributes to the antiviral activity of ribavirin. Therefore, the antiviral activities of (i) ribavirin, (ii) its 5-ethynyl analogue, 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR), and (iii) mycophenolic acid (MPA) (a compound that inhibits only cellular IMP dehydrogenase activity) were studied on the replication of flaviviruses and paramyxoviruses. In addition, the effects of these three compounds on intracellular GTP pools were assessed. A linear correlation was observed over a broad concentration range between the antiviral activities of ribavirin, EICAR, and MPA and the effects of these compounds on GTP pool depletion. When the 50% effective concentrations (EC50s) for the antiviral activities of ribavirin, EICAR, and MPA were plotted against the respective EC50 values for GTP pool depletion, a linear correlation was calculated. These data provide compelling evidence that the predominant mechanism of action of ribavirin in vitro against flavi- and paramyxoviruses is based on inhibition of cellular IMP dehydrogenase activity.