The heptauridine tract at each gene end and intergenic region (IGR) at the gene junctions of vesicular stomatitis virus (VSV) have effects on synthesis of the downstream mRNA, independent of their respective roles in termination of the upstream mRNA. To investigate the role of the U tract and the IGR in downstream gene transcription, we altered the N/P gene junction of infectious VSV such that transcription levels would be affected and result in altered molar ratios of the N and P proteins, which are critical for optimal viral RNA replication. The changes included extended IGRs between the N and P genes and shortening the length of the heptauridine tract upstream of the P gene start. Viruses having various combinations of these changes were recovered from cDNA and selective pressure for efficient viral replication was applied by sequential passage in cell culture. The replicative ability and sequence at the altered intergenic junctions were monitored throughout the passages to compare the effects of the changes at the IGR and U tract. VSV variants with wild-type U tracts upstream of the P gene replicated to levels similar to wt VSV. Variants with shortened U tracts were reduced in their ability to replicate. With passage, populations emerged that replicated to higher levels. Sequence analysis revealed that mutations had been selected for in these populations that increased the length of the U tract. This correlated with an increase in abundance of P mRNA and protein to provide improved N:P protein molar ratios. Extended IGRs resulted in decreased downstream transcription but the effect was not as extensive as that caused by shortened U tracts. Extended IGRs were not selected against in 5 passages. Our results indicate that the size of the upstream gene end U tract is an important determinant of efficient downstream gene transcription in infectious virus.