Abstract Small leader RNAs, copied from the extreme 3′ ends of the minus and plus strands of the vesicular stomatitis virus (VSV) genome, are thought to play a central role in the regulation of viral transcription and replication. We describe here a novel class of VSV mutants, denoted pol R, in which termination at leader sites in vitro is. specifically suppressed. We have assayed for the presence of leader RNAs and readthrough transcripts in reaction products from standard virion templates (plus leader) and defective interfering particle templates (minus leader). In both cases, mutant virions gave rise to a much higher proportion of readthrough transcripts than wild type (>80% vs ∼10%). Reconstitution experiments with separated ribonucleoprotein (RNP) templates and polymerase protein fractions revealed, surprisingly, that the N protein moiety of the RNP template was responsible for readthrough. This conclusion was further supported by protein analyses that showed a similar charge change in the N protein of two independently isolated pol R VSV mutants. These results lead us to propose that modification of the N protein may regulate termination at leader RNA sites.