We have analyzed the process of partial transcription termination (attenuation), which results in nonequimolar synthesis of vesicular stomatitis virus (VSV) mRNAs during sequential transcription. Comparison of the level of transcription of defined regions of the VSV genome by DNA-RNA hybridization shows that attenuation occurs at or near the intergenic regions, rather than nonspecifically throughout the genome. Transcription decreases 29-33% across the junctions of the N-NS, NS-M and M-G genes, resulting in a cumulative effect on gene expression. This is the first example of a site-specific attenuation mechanism in a eucaryotic system. Analysis of the kinetics of transcription in vitro shows that transcription appears to be discontinuous, with significant pauses (2.5-5.7 min) occurring at or near the intergenic regions. Such pauses may occur during polyadenylation by a "slippage" mechanism at the U7 sequences present at each gene junction, or may be due to some other process, such as initiation or capping, which is slow relative to transcription.