Here we report the development of two independent assays which demonstrate for the first time that exogenous model RNA templates based on influenza virus virion RNA (vRNA) are transcribed in vitro to produce polyadenylated mRNA. We investigated the activities of mutated templates with known polymerase binding properties to test our model that polyadenylation occurs when a polymerase complex, which is bound to conserved 5' sequences of vRNA, prevents read-through of the U track at which polyadenylation subsequently occurs by reiterative copying. Mutated templates with perturbed polymerase binding sites (i.e., a deletion mutant lacking the first 4 5' residues and a U-->A point mutant at the third residue) initiated transcription in the in vitro assay but failed to produce polyadenylated transcripts, whereas an A-->U point mutant at the fourth residue, which retained polymerase binding properties similar to those of the wild type, produced polyadenylated transcripts. Our results show that nucleotides within the conserved 5' sequence are required for polyadenylation and support the hypothesis that polymerase binding to 5' sequences of the template is required for mRNA synthesis.