The nonsegmented negative-strand RNA (NNS) viruses have a single-stranded RNA genome tightly encapsidated by the viral nucleocapsid protein. The viral polymerase transcribes the genome responding to specific gene-start and gene-end sequences to yield a series of discrete monocistronic mRNAs. These mRNAs are not produced in equimolar amounts; rather, their abundance reflects the position of the gene with respect to the single 3'-proximal polymerase entry site. Promoter-proximal genes are transcribed in greater abundance than more distal genes due to a localized transcriptional attenuation at each gene junction. In recent years, the application of reverse genetics to the NNS viruses has allowed an examination of the role of the gene-start and gene-end sequences in regulating mRNA synthesis. These studies have defined specific sequences required for initiation, 5' modification, termination, and polyadenylation of the viral mRNAs. In the present report, working with Vesicular stomatitis virus, the prototypic Rhabdovirus, we demonstrate that a gene-end sequence must be positioned a minimal distance from a gene-start sequence for the polymerase to efficiently terminate transcription. Gene-end sequences were almost completely ignored in transcriptional units less than 51 nucleotides. Transcriptional units of 51 to 64 nucleotides allowed termination at the gene-end sequence, although the frequency with which polymerase failed to terminate and instead read through the gene-end sequence to generate a bicistronic transcript was enhanced compared to the observed 1 to 3% for wild-type viral mRNAs. In all instances, failure to terminate at the gene end prevented initiation at the downstream gene start site. In contrast to this size requirement, we show that the sequence between the gene-start and gene-end signals, or its potential to adopt an RNA secondary structure, had only a minor effect on the efficiency with which polymerase terminated transcription. We suggest three possible explanations for the failure of polymerase to terminate transcription in response to a gene-end sequence positioned close to a gene-start sequence which contribute to our emerging picture of the mechanism of transcriptional regulation in this group of viruses.