Infection of mouse myeloma cells (MPC-11) with vesicular stomatitis (VS) virus resulted in rapid and marked reduction in cellular RNA synthesis considerably before cell viability was compromised. Mouse myeloma cells responded maximally to viral infection at a multiplicity of 1 and were considerably more se;sitive to shut-off of RNA synthesis than were mouse L cells or BHK-21 cells. This inhibition of cellular RNA synthesis was shown not to be caused by differential membrane permeability of infected and uninfected MPC-11 cells to [3H]uridine, nor was it due to greater degradation of previously synthesized RNA. VS viral infection appeared not to impede transport of newly synthesized nuclear RNA to the cytoplasm; moreover, infected cells accumulated polyadenylated mRNA at the same rate as did uninfected cells. Polyacrylamide gel electrophoresis of newly synthesized nuclear RNA demonstrated that the polydisperse nature and size distribution were not affected by VS viral infection. Isolated nuclei of infected MPC-11 cells also inhibited greatly impaired capacity to synthesize RNA despite the absence of cytoplasmic factors. Infected-cell cytosol did not inhibit transcription by uninfected-cell nuclei, nor did uninfected-cell cytosol reverse viral inhibition of nuclear transcription. Studies with alpha-amanitin revealed that VS viral infection inhibited the activity of polymerases I, II, and III, but only polymerase II was affected progressively throughout infection and to a much greater extent. These data suggest that, even at low multiplicities of infection, VS virus rapidly shuts off cellular RNA synthesis at the level of nuclear transcription.