In vitro RNA synthesis by tobacco mosaic virus and cowpea chlorotic mottle virus replicase were inhibited by cordycepin triphosphate. Inhibition could be overcome with higher concentrations of ATP in assay mixtures but not with UTP. Products synthesized in vitro by tobacco mosaic virus RNA replicase in the presence of inhibitor revealed replicative form but not replicative intermediate RNAs. These results suggest that cordycepin triphosphate competes specifically with ATP and results in premature termination of viral RNA synthesis in vitro.