The effect of phosphorylated ribavirin on a vesicular stomatitis virus (VSV) in vitro transcription reaction was examined. Viral mRNA synthesized in the presence of the 5' mono-, di-, and triphosphorylated forms of the drug translated with equal efficiencies under the test conditions. However, all three phosphorylated species inhibited VSV transcription. The mono- and diphosphorylated forms of the drug possessed approximately two to three times the inhibitory activity as the triphosphorylated form. Transcripts synthesized in the presence of drug were full length and were absent of incorporated drug. Inhibition by ribavirin 5'-diphosphate could be reversed by the addition of UTP, CTP, and GTP, while the addition of GDP to the reaction did not reverse inhibition. Ribavirin diphosphate was added to a La Crosse virus in vitro transcription assay to determine whether an inhibitory effect could be established in a viral system that was more sensitive to ribavirin than was VSV; it led to profound inhibition of RNA synthesis at concentrations as low as 0.1 microgram/ml. These data suggest that ribavirin has an effect on the initial steps of transcription by some RNA-dependent RNA polymerases and that this effect may be mediated by several phosphorylated forms.