The influenza virus vRNA promoter was characterized: a complete set of single substitution mutants was generated in the fifteen 3' terminal nucleotides of a synthetic model RNA containing the reporter gene chloramphenicol acetyl transferase (CAT). The contribution of each nucleotide to the function of the promoter was tested by an in vitro assay. This system involves reconstitution of template (mutant) RNAs and purified viral polymerase; the system is primer-dependent and yields full-length complementary (c)RNA and not poly A-containing mRNA. The results of this in vitro replication assay suggest that (1) nucleotides 1 to 14 at the 3' terminus comprise the promoter sequence of the vRNA, (2) not all the mutations in the first 14 nucleotides affect vRNA promoter activity equally and (3) changes in positions 2 and 11 have the greatest effect on this promoter activity. In addition, the template (mutant) RNAs were examined in an in vivo assay. This system involves transfection of plasmid DNA-derived template (mutant) RNAs into helper virus-infected cells and measurement of levels of CAT activity. The expression of template RNAs was found to be highly sensitive to mutations in almost any of the first 14 positions. Differences in the results of the in vivo and the in vitro system are possibly due to the presence of overlapping cis-acting signals which are required for replication of vRNA and the expression of mRNA. Deletion and addition of nucleotides at the 3' end of the promoter resulted in a drastic reduction in template activity in both the in vitro and in vivo assays.